Microorganisms and methods for production of specific length fatty alcohols and related compounds

ABSTRACT

The invention provides non-naturally occurring microbial organisms containing a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the microbial organisms selectively produce a fatty alcohol, fatty aldehyde or fatty acid of a specified length. Also provided are non-naturally occurring microbial organisms having a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the microbial organisms further include an acetyl-CoA pathway. In some aspects, the microbial organisms of the invention have select gene disruptions or enzyme attenuations that increase production of fatty alcohols, fatty aldehydes or fatty acids. The invention additionally provides methods of using the above microbial organisms to produce a fatty alcohol, a fatty aldehyde or a fatty acid.

This application claims the benefit of priority of U.S. Provisionalapplication Ser. No. 61/714,144, filed Oct. 15, 2012, the entirecontents of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes, andmore specifically to organisms having specific length fatty alcohol,fatty aldehyde or fatty acid biosynthetic capacity.

Primary alcohols are a product class of compounds having a variety ofindustrial applications which include a variety of biofuels andspecialty chemicals. Primary alcohols also can be used to make a largenumber of additional industrial products including polymers andsurfactants. For example, higher primary alcohols, also known as fattyalcohols (C₄-C₂₄) and their ethoxylates are used as surfactants in manyconsumer detergents, cleaning products and personal care productsworldwide such as laundry powders and liquids, dishwashing liquid andhard surface cleaners. They are also used in the manufacture of avariety of industrial chemicals and in lubricating oil additives.Specific length fatty alcohols, such as octanol and hexanol, have usefulorganoleptic properties and have long been employed as fragrance andflavor materials. Smaller chain length C₄-C₈ alcohols (e.g., butanol)are used as chemical intermediates for production of derivatives such asacrylates used in paints, coatings, and adhesives applications.

Fatty alcohols are currently produced from, for example, hydrogenationof fatty acids, hydroformylation of terminal olefins, partial oxidationof n-paraffins and the Al-catalyzed polymerization of ethylene.Unfortunately, it is not commercially viable to produce fatty alcoholsdirectly from the oxidation of petroleum-based linear hydrocarbons(n-paraffins). This impracticality is because the oxidation ofn-paraffins produces primarily secondary alcohols, tertiary alcohols orketones, or a mixture of these compounds, but does not produce highyields of fatty alcohols. Additionally, currently known methods forproducing fatty alcohols suffer from the disadvantage that they arerestricted to feedstock which is relatively expensive, notably ethylene,which is produced via the thermal cracking of petroleum. In addition,current methods require several steps, and several catalyst types.

Fatty alcohol production by microorganisms involves fatty acid synthesisfollowed by acyl-reduction steps. The universal fatty acid biosynthesispathway found in most cells has been investigated for production offatty alcohols and other fatty acid derivatives. There is currently agreat deal of improvement that can be achieved to provide more efficientbiosynthesis pathways for fatty alcohol production with significantlyhigher theoretical product and energy yields.

Thus, there exists a need for alternative means for effectivelyproducing commercial quantities of fatty alcohols. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF INVENTION

The invention provides non-naturally occurring microbial organismscontaining fatty alcohol, fatty aldehyde or fatty acid pathways. In someembodiments, the non-naturally occurring microbial organism of theinvention has a malonyl-CoA independent fatty acyl-CoA elongation(MI-FAE) cycle and/or a malonyl-CoA dependent fatty acyl-CoA elongation(MD-FAE) cycle in combination with a termination pathway as depicted inFIGS. 1, 6 and 7, wherein an enzyme of the MI-FAE cycle, MD-FAE cycle ortermination pathway is encoded by at least one exogenous nucleic acidand is expressed in a sufficient amount to produce a fatty alcohol,fatty aldehyde or fatty acid of Formula (I):

wherein R₁ is C₁₋₂₄ linear alkyl; R₂ is CH₂OH, CHO, or COOH; R₃ is H,OH, or oxo (═O); and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four, wherein the substrateof each of said enzymes of the MI-FAE cycle, the MD-FAE cycle and thetermination pathway are independently selected from a compound ofFormula (II), malonyl-CoA, propionyl-CoA or acetyl-CoA:

wherein R₁ is C₁₋₂₄ linear alkyl; R₃ is H, OH, or oxo (═O); R₄ is S-CoA,ACP, OH or H; and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four; wherein said one ormore enzymes of the MI-FAE cycle are each selective for a compound ofFormula (II) having a number of carbon atoms at R₁ that is no greaterthan the number of carbon atoms at R₁ of said compound of Formula (I),wherein said one or more enzymes of the MD-FAE cycle are each selectivefor a compound of Formula (II) having a number of carbon atoms at R₁that is no greater than the number of carbon atoms at R₁ of saidcompound of Formula (I), and wherein said one or more enzymes of thetermination pathway are each selective for a compound of Formula (II)having a number of carbon atoms at R₁ that is no less than the number ofcarbon atoms at R₁ of said compound of Formula (I).

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a fatty alcohol, fatty aldehyde or fatty acidpathway, wherein the microbial organism further includes an acetyl-CoApathway and at least one exogenous nucleic acid encoding an acetyl-CoApathway enzyme expressed in a sufficient amount to produce acetyl-CoA,wherein the acetyl-CoA pathway includes a pathway shown in FIG. 2, 3, 4or 5.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a fatty alcohol, fatty aldehyde or fatty acidpathway, wherein the microbial organism has one or more genedisruptions, wherein the one or more gene disruptions occur inendogenous genes encoding proteins or enzymes involved in: nativeproduction of ethanol, glycerol, acetate, formate, lactate, CO₂, fattyacids, or malonyl-CoA by said microbial organism; transfer of pathwayintermediates to cellular compartments other than the cytosol; or nativedegradation of a MI-FAE cycle intermediate, MD-FAE cycle intermediate ora termination pathway intermediate by the microbial organism, the one ormore gene disruptions confer increased production of a fatty alcohol,fatty aldehyde or fatty acid in the microbial organism.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a fatty alcohol, fatty aldehyde or fatty acidpathway, wherein one or more enzymes of the MI-FAE cycle, MD-FAE cycleor the termination pathway preferentially react with an NADH cofactor orhave reduced preference for reacting with an NAD(P)H cofactor.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a fatty alcohol, fatty aldehyde or fatty acidpathway, wherein the microbial organism has one or more gene disruptionsin genes encoding proteins or enzymes that result in an increased ratioof NAD(P)H to NAD(P) present in the cytosol of the microbial organismfollowing the disruptions.

In some embodiments, the non-naturally occurring microbial organism ofthe invention is Crabtree positive and is in culture medium comprisingexcess glucose. In such conditions, as described herein, the microbialorganism can result in increasing the ratio of NAD(P)H to NAD(P) presentin the cytosol of the microbial organism.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a fatty alcohol, fatty aldehyde or fatty acidpathway, wherein the microbial organism has at least one exogenousnucleic acid encoding an extracellular transporter or an extracellulartransport system for a fatty alcohol, fatty aldehyde or fatty acid ofthe invention.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a fatty alcohol, fatty aldehyde or fatty acidpathway, wherein the microbial organism one or more endogenous enzymesinvolved in: native production of ethanol, glycerol, acetate, formate,lactate, CO₂, fatty acids, or malonyl-CoA by said microbial organism;transfer of pathway intermediates to cellular compartments other thanthe cytosol; or native degradation of a MI-FAE cycle intermediate, aMD-FAE cycle intermediate or a termination pathway intermediate by saidmicrobial organism, has attenuated enzyme activity or expression levels.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a fatty alcohol, fatty aldehyde or fatty acidpathway, wherein the microbial organism has attenuated enzyme activityor expression levels for one or more endogenous enzymes involved in theoxidation of NAD(P)H or NADH.

The invention additionally provides methods of using the above microbialorganisms to produce a fatty alcohol, a fatty aldehyde or a fatty acidby culturing a non-naturally occurring microbial organism containing afatty alcohol, fatty aldehyde or fatty acid pathway under conditions andfor a sufficient period of time to produce a fatty alcohol, fattyaldehyde or fatty acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary MI-FAE cycle and/or MD-FAE cycle incombination with termination pathways for production of fatty alcohols,aldehydes, or acids from the acyl-CoA intermediate of the MI-FAE cycleor MD-FAE cycle. Enzymes are: A. Thiolase; B. 3-Oxoacyl-CoA reductase;C. 3-Hydroxyacyl-CoA dehydratase; D. Enoyl-CoA reductase; E. Acyl-CoAreductase (aldehyde forming); F. Alcohol dehydrogenase; G. Acyl-CoAreductase (alcohol forming); H. acyl-CoA hydrolase, transferase orsynthase; J. Acyl-ACP reductase; K. Acyl-CoA:ACP acyltransferase; L.Thioesterase; N. Aldehyde dehydrogenase (acid forming) or carboxylicacid reductase; and O. Elongase.

FIG. 2 shows exemplary pathways for production of cytosolic acetyl-CoAfrom pyruvate or threonine. Enzymes are: A. pyruvate oxidase(acetate-forming); B. acetyl-CoA synthetase, ligase or transferase; C.acetate kinase; D. phosphotransacetylase; E. pyruvate decarboxylase; F.acetaldehyde dehydrogenase; G. pyruvate oxidase (acetyl-phosphateforming); H. pyruvate dehydrogenase, pyruvate:ferredoxin oxidoreductase,pyruvate:NAD(P)H oxidoreductase or pyruvate formate lyase; I.acetaldehyde dehydrogenase (acylating); and J. threonine aldolase.

FIG. 3 shows exemplary pathways for production of acetyl-CoA fromphosphoenolpyruvate (PEP). Enzymes are: A. PEP carboxylase or PEPcarboxykinase; B. oxaloacetate decarboxylase; C. malonate semialdehydedehydrogenase (acetylating); D. acetyl-CoA carboxylase or malonyl-CoAdecarboxylase; F. oxaloacetate dehydrogenase or oxaloacetateoxidoreductase; G. malonate semialdehyde dehydrogenase (acylating); H.pyruvate carboxylase; J. malonate semialdehyde dehydrogenase; K.malonyl-CoA synthetase or transferase; L. malic enzyme; M. malatedehydrogenase or oxidoreductase; and N. pyruvate kinase or PEPphosphatase.

FIG. 4 shows exemplary pathways for production of cytosolic acetyl-CoAfrom mitochondrial acetyl-CoA using citrate and malate transporters.Enzymes are: A. citrate synthase; B. citrate transporter; C.citrate/malate transporter; D. ATP citrate lyase; E. citrate lyase; F.acetyl-CoA synthetase or transferase; H. cytosolic malate dehydrogenase;I. malate transporter; J. mitochondrial malate dehydrogenase; K. acetatekinase; and L. phosphotransacetylase.

FIG. 5 shows exemplary pathways for production of cytosolic acetyl-CoAfrom mitochondrial acetyl-CoA using citrate and oxaloacetatetransporters. Enzymes are: A. citrate synthase; B. citrate transporter;C. citrate/oxaloacetate transporter; D. ATP citrate lyase; E. citratelyase; F. acetyl-CoA synthetase or transferase; G) oxaloacetatetransporter; K) acetate kinase; and L) phosphotransacetylase.

FIG. 6 shows an exemplary MI-FAE cycle and/or MD-FAE cycle forelongating the linear alkyl of R₁. Enzymes are: A. Thiolase; B.3-Ketoacyl-CoA reductase; C. 3-Hydroxyacyl-CoA dehydratase; D. Enoyl-CoAreductase; and E. Elongase.

FIG. 7 shows an exemplary termination cycle for generating a fattyalcohol, fatty aldehyde or fatty acid from any of the MI-FAE cycleintermediates or MD-FAE cycle intermediates of FIG. 6. Enzymes are: E.MI-FAE/MD-FAE intermediate-CoA reductase (aldehyde forming); F. Alcoholdehydrogenase; G. MI-FAE/MD-FAE intermediate-CoA reductase (alcoholforming); H. MI-FAE/MD-FAE intermediate-CoA hydrolase, transferase orsynthase; J. MI-FAE/MD-FAE intermediate-ACP reductase; K. MI-FAE/MD-FAEintermediate-CoA:ACP acyltransferase; L. Thioesterase; and N. Aldehydedehydrogenase (acid forming) or carboxylic acid reductase. R1 is C1-24linear alkyl; R₃ is H, OH, or oxo (═O) and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four.

FIG. 8 shows exemplary compounds that can be produced from the fourMI-FAE or MD-FAE cycle intermediates using the cycles depicted in FIG. 6and the termination pathways depicted in FIG. 7. R is C₁₋₂₄ linearalkyl.

FIG. 9 depicts the production of 1,3-butanediol (FIG. 9A) or ethanol(FIG. 9B) in S. cerevisiae transformed with plasmids comprising genesencoding various MI-FAE cycle and termination pathway enzymes, eitherwith or without pflAV or PDH bypass, as provided in Example X.

FIG. 10 depicts the production of pyruvic acid (FIG. 10A), succinic acid(FIG. 12B), acetic acid (FIG. 12C) or glucose (FIG. 12D) in S.cerevisiae transformed with plasmids comprising genes encoding variousMI-FAE cycle and termination pathway enzymes, either with or withoutpflAV or PDH bypass, as provided in Example X.

FIG. 11 depicts the production of 1,3-butanediol in S. cerevisiaetransformed with plasmids comprising genes encoding various MI-FAE cycleand termination pathway enzymes, either with or without pflAV or PDHbypass, as provided in Example X.

FIG. 12 depicts the estimated specific activity of five thiolases foracetyl-CoA condensation activity in E. coli as provided in Example XI.

FIG. 13 depicts the estimated specific activity of two thiolases (1491and 560) cloned in dual promoter yeast vectors with 1495 (a3-hydroxybutyryl-CoA dehydrogenase) for acetyl-CoA condensation activityin E. coli as provided in Example XI.

FIG. 14 depicts the time course of fluorescence detection of oxidationof NADH, which is used to measure the metabolism of acetoacetyl-CoA to3-hydroxybutyryl-CoA by 3-hydroxybutyryl-CoA dehydrogenase, as providedin Example XI. Acetoacetyl-CoA is metabolized to 3-hydroxybutyryl-CoA by3-hydroxybutyryl-CoA dehydrogenase. The reaction requires oxidation ofNADH, which can be monitored by fluorescence at an excitation wavelengthat 340 nm and an emission at 460 nm. The oxidized form, NAD+, does notfluoresce. 1495, the Hbd from Clostridium beijerinckii, was assayed inthe dual promoter yeast vectors that contained either 1491 (vectorid=pY3Hd17) or 560 (vector id=pY3Hd16).

FIG. 15 depicts levels of NAD(P)H oxidation in the presence of 1 or 5ug/ml NADH or 1 or 5 ug/ml NADPH, and shows that the Hbd prefers NADHover NADPH, as provided in Example XI.

FIG. 16 depicts the activity data for crude lysates of an aldehydereductase that converts 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehydeand requires NAD(P)H oxidation, which can be used to monitor enzymeactivity, as provided in Example XI. The Ald from Lactobacillus brevis(Gene ID 707) was cloned in a dual vector that contained the alcoholdehydrogenase from Clostridium saccharoperbutylacetonicum (Gene ID 28).These two enzymes were cloned in another dual promoter yeast vectorcontaining a Leu marker. A 707 lysate from E. coli was used as astandard.

FIG. 17 depicts the evaluation of ADH (Gene 28) in the dual promotervector with ALD (Gene 707) with butyraldehyde, a surrogate substrate for3-hydroxybutyraldehyde. 1,3-BDO is formed by an alcohol dehydrogenase(Adh), which reduces 3-hydroxybutyraldehyde in the presence of NAD(P)H,and the oxidation of NAD(P)H is used to monitor the reaction.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a fattyalcohol, fatty aldehyde or fatty alcohol biosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides, or functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” are intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “ACP” or “acyl carrier protein” refers to anyof the relatively small acidic proteins that are associated with thefatty acid synthase system of many organisms, from bacteria to plants.ACPs can contain one 4′-phosphopantetheine prosthetic group boundcovalently by a phosphate ester bond to the hydroxyl group of a serineresidue. The sulfhydryl group of the 4′-phosphopantetheine moiety servesas an anchor to which acyl intermediates are (thio)esterified duringfatty-acid synthesis. An example of an ACP is Escherichia coli ACP, aseparate single protein, containing 77 amino-acid residues (8.85 kDa),wherein the phosphopantetheine group is linked to serine 36.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acids refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

As used herein, the term “gene disruption,” or grammatical equivalentsthereof, is intended to mean a genetic alteration that renders theencoded gene product inactive or attenuated. The genetic alteration canbe, for example, deletion of the entire gene, deletion of a regulatorysequence required for transcription or translation, deletion of aportion of the gene which results in a truncated gene product, or by anyof various mutation strategies that inactivate or attenuate the encodedgene product. One particularly useful method of gene disruption iscomplete gene deletion because it reduces or eliminates the occurrenceof genetic reversions in the non-naturally occurring microorganisms ofthe invention. A gene disruption also includes a null mutation, whichrefers to a mutation within a gene or a region containing a gene thatresults in the gene not being transcribed into RNA and/or translatedinto a functional gene product. Such a null mutation can arise from manytypes of mutations including, for example, inactivating point mutations,deletion of a portion of a gene, entire gene deletions, or deletion ofchromosomal segments.

As used herein, the term “growth-coupled” when used in reference to theproduction of a biochemical product is intended to mean that thebiosynthesis of the referenced biochemical product is produced duringthe growth phase of a microorganism. In a particular embodiment, thegrowth-coupled production can be obligatory, meaning that thebiosynthesis of the referenced biochemical is an obligatory productproduced during the growth phase of a microorganism.

As used herein, the term “attenuate,” or grammatical equivalentsthereof, is intended to mean to weaken, reduce or diminish the activityor amount of an enzyme or protein. Attenuation of the activity or amountof an enzyme or protein can mimic complete disruption if the attenuationcauses the activity or amount to fall below a critical level requiredfor a given pathway to function. However, the attenuation of theactivity or amount of an enzyme or protein that mimics completedisruption for one pathway, can still be sufficient for a separatepathway to continue to function. For example, attenuation of anendogenous enzyme or protein can be sufficient to mimic the completedisruption of the same enzyme or protein for production of a fattyalcohol, fatty aldehyde or fatty acid product of the invention, but theremaining activity or amount of enzyme or protein can still besufficient to maintain other pathways, such as a pathway that iscritical for the host microbial organism to survive, reproduce or grow.Attenuation of an enzyme or protein can also be weakening, reducing ordiminishing the activity or amount of the enzyme or protein in an amountthat is sufficient to increase yield of a fatty alcohol, fatty aldehydeor fatty acid product of the invention, but does not necessarily mimiccomplete disruption of the enzyme or protein.

The term “fatty alcohol,” as used herein, is intended to mean analiphatic compound that contains one or more hydroxyl groups andcontains a chain of 4 or more carbon atoms. The fatty alcohol possessesthe group —CH₂OH that can be oxidized so as to form a correspondingaldehyde or acid having the same number of carbon atoms. A fatty alcoholcan also be a saturated fatty alcohol, an unsaturated fatty alcohol, a1,3-diol, or a 3-oxo-alkan-1-ol. Exemplary fatty alcohols include acompound of Formula (III)-(VI):

wherein R₁ is a C₁₋₂₄ linear alkyl.

The term “fatty aldehyde,” as used herein, is intended to mean analiphatic compound that contains an aldehyde (CHO) group and contains achain of 4 or more carbon atoms. The fatty aldehyde can be reduced toform the corresponding alcohol or oxidized to form the carboxylic acidhaving the same number of carbon atoms. A fatty aldehyde can also be asaturated fatty aldehyde, an unsaturated fatty aldehyde, a3-hydroxyaldehyde or 3-oxoaldehyde. Exemplary fatty aldehydes include acompound of Formula (VII)-(X):

wherein R₁ is a C₁₋₂₄ linear alkyl.

The term “fatty acid,” as used herein, is intended to mean an aliphaticcompound that contains a carboxylic acid group and contains a chain of 4or more carbon atoms. The fatty acid can be reduced to form thecorresponding alcohol or aldehyde having the same number of carbonatoms. A fatty acid can also be a saturated fatty acid, an unsaturatedfatty acid, a 3-hydroxyacid or a 3-oxoacids. Exemplary fatty acidsinclude a compound of Formula (XI)-(XIV):

wherein R₁ is a C₁₋₂₄ linear alkyl.

The term “alkyl” refers to a linear saturated monovalent hydrocarbon.The alkyl can be a linear saturated monovalent hydrocarbon that has 1 to24 (C₁₋₂₄), 1 to 17 (C₁₋₁₇), or 9 to 13 (C₉₋₁₃) carbon atoms. Examplesof alkyl groups include, but are not limited to, methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl.For example, C₉₋₁₃ alkyl refers to a linear saturated monovalenthydrocarbon of 9 to 13 carbon atoms.

The invention disclosed herein is based, at least in part, onrecombinant microorganisms capable of synthesizing fatty alcohols, fattyaldehydes, or fatty acids using a malonyl-CoA-independent fatty acidelongation (MI-FAE) cycle and/or malonyl-CoA dependent fatty acidelongation cycle (MD-FAE) cycle in combination with a terminationpathway. In some embodiments, the microorganisms of the invention canutilize a heterologous MI-FAE cycle and/or a MD-FAE cycle coupled withan acyl-CoA termination pathway to form fatty alcohols, fatty aldehydes,or fatty acids. The MI-FAE cycle can include a thiolase, a 3-oxoacyl-CoAreductase, a 3-hydroxyacyl-CoA dehydratase and an enoyl-CoA reductase.The MD-FAE cycle can include an elongase, a 3-oxoacyl-CoA reductase, a3-hydroxyacyl-CoA dehydratase and an enoyl-CoA reductase. Each passagethrough the MI-FAE cycle and/or the MD-FAE cycle results in theformation of an acyl-CoA elongated by a single two carbon unit comparedto the acyl-CoA substrate entering the elongation cycle. Products can beeven or odd chain length, depending on the initial substrate enteringthe acyl-CoA elongation pathway, i.e. two acety-CoA substrates,malonyl-CoA or one acetyl-CoA substrate combined with a propionyl-CoAsubstrate. Elongation of the two acetyl-CoA substrates or malonyl-CoAproduces an even chain length product, whereas elongation with thepropionyl-CoA substrate produces an odd chain length product. Atermination pathway catalyzes the conversion of a MI-FAE intermediateand/or a MD-FAE intermediate, such as the acyl-CoA, to its correspondingfatty alcohol, fatty aldehyde, or fatty acid product. MI-FAE cycle,MD-FAE cycle and termination pathway enzymes can be expressed in one ormore compartments of the microorganism. For example, in one embodiment,all MI-FAE cycle and termination pathway enzymes are expressed in thecytosol. In another embodiment, all MD-FAE cycle and termination pathwayenzymes are expressed in the cytosol. Additionally, the microorganismsof the invention can be engineered to optionally secret the desiredproduct into the culture media or fermentation broth for furthermanipulation or isolation.

Products of the invention include fatty alcohols, fatty aldehydes, orfatty acids derived from intermediates of the MI-FAE cycle and/or MD-FAEcycle. For example, alcohol products can include saturated fattyalcohols, unsaturated fatty alcohols, 1,3-diols, and 3-oxo-alkan-1-ols.Aldehyde products can include saturated fatty aldehydes, unsaturatedfatty aldehydes, 3-hydroxyaldehydes and 3-oxoaldehydes. Acid productscan include saturated fatty acids, unsaturated fatty acids,3-hydroxyacids and 3-oxoacids. These products can further be convertedto derivatives such as fatty esters, either by chemical or enzymaticmeans. Methods for converting fatty alcohols to esters are well known inthe art.

The invention also encompasses fatty alcohol, fatty aldehyde, and fattyacid chain-length control strategies in conjunction with host strainengineering strategies, such that the non-naturally occurringmicroorganism of the invention efficiently directs carbon and reducingequivalents toward fermentation products of a specific chain length.

Recombinant microorganisms of the invention can produce commercialquantities of a fatty alcohol, fatty aldehyde, or fatty acid ranging inchain length from four carbon atoms (C₄) to twenty-four carbon atoms(C₂₄) or more carbon atoms. The microorganism of the invention canproduce a desired product that is at least 50%, 60%, 70%, 75%, 85%, 90%,95% or more selective for a particular chain length. The carbonchain-length of the product is controlled by one or more enzymes of theMI-FAE cycle (steps A/B/C/D of FIG. 6) and/or one or more enzymes of theMD-FAE cycle (steps E/B/C/D of FIG. 6) in combination with one or moretermination pathway enzymes (steps E-N of FIG. 7). Chain length can becapped during the elongation cycle by one or more MI-FAE cycle enzymes(thiolase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and/orenoyl-CoA reductase) exhibiting selectivity for MI-FAE cycle substrateshaving a number of carbon atoms that are no greater than the desiredproduct size. Alternatively, or in addition, chain length can be cappedduring the elongation cycle by one or more MD-FAE cycle enzymes(elongase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and/orenoyl-CoA reductase). Chain length can be further constrained by one ormore enzymes catalyzing the conversion of the MI-FAE cycle intermediateto the fatty alcohol, fatty aldehyde or fatty acid product such that theone or more termination enzymes only reacts with substrates having anumber of carbon atoms that are no less than the desired fatty alcohol,fatty aldehyde or fatty acid product.

The termination pathway enzymes catalyzing conversion of a MI-FAE-CoAintermediate or MD-FAE-CoA intermediate to a fatty alcohol can includecombinations of a fatty acyl-CoA reductase (alcohol or aldehydeforming), a fatty aldehyde reductase, an acyl-ACP reductase, anacyl-CoA:ACP acyltransferase, a thioesterase, an acyl-CoA hydrolaseand/or a carboxylic acid reductase (pathways G; E/F; K/J/F; H/N/F; orK/L/N/F of FIG. 7). Termination pathway enzymes for converting aMI-FAE-CoA intermediate or MD-FAE-CoA intermediate to a fatty acid caninclude combinations of a thioesterase, a CoA hydrolase, an acyl-CoA:ACPacyltransferase, an aldehyde dehydrogenase and/or an acyl-ACP reductase(pathways H; K/L; E/N; K/J/N of FIG. 7). For production of a fattyaldehyde, the termination pathway enzymes can include combinations of afatty acyl-CoA reductase (aldehyde forming), an acyl-ACP reductase, anacyl-CoA:ACP acyltransferase, a thioesterase, an acyl-CoA hydrolaseand/or a carboxylic acid reductase (pathways E; K/J; H/N; or K/L/N ofFIG. 7).

The non-naturally occurring microbial organisms of the invention canalso efficiently direct cellular resources, including carbon, energy andreducing equivalents, to the production of fatty alcohols, fattyaldehydes and fatty acids, thereby resulting in improved yield,productivity and/or titer relative to a naturally occurring organism. Inone embodiment, the microorganism is modified to increase cytosolicacetyl-CoA levels. In another embodiment, the microorganism is modifiedto efficiently direct cytosolic acyl-CoA into fatty alcohols, fattyaldehydes or fatty acids rather than other byproducts or cellularprocesses. Enzymes or pathways that lead to the formation of byproductscan be attenuated or deleted. Exemplary byproducts include, but are notlimited to, ethanol, glycerol, lactate, acetate, esters and carbondioxide. Additional byproducts can include fatty-acyl-CoA derivativessuch as alcohols, alkenes, alkanes, esters, acids and aldehydes.Accordingly, a byproduct can include any fermentation product divertingcarbon and/or reducing equivalents from the product of interest.

In another embodiment, the availability of reducing equivalents or redoxratio is increased. In yet another embodiment, the cofactor requirementsof the microorganism are balanced such that the same reduced cofactorsgenerated during carbon assimilation and central metabolism are utilizedby MI-FAE cycle, MD-FAE cycle and/or termination pathway enzymes. In yetanother embodiment, the fatty alcohol, fatty aldehyde or fatty acidproducing organism expresses a transporter which exports the fattyalcohol, fatty aldehyde or fatty acid from the cell.

Microbial organisms capable of fatty alcohol production are exemplifiedherein with reference to the Saccharomyces cerevisaie geneticbackground. However, with the complete genome sequence available now forthousands of species (with more than half of these available on publicdatabases such as the NCBI), the identification of an alternate specieshomolog for one or more genes, including for example, orthologs,paralogs and nonorthologous gene displacements, and the interchange ofgenetic alterations between eukaryotic organisms is routine and wellknown in the art. Accordingly, the metabolic alterations enablingproduction of fatty alcohols described herein with reference to aparticular organism such as Saccharomyces cerevisiae can be readilyapplied to other microorganisms. Given the teachings and guidanceprovided herein, those skilled in the art understand that a metabolicalteration exemplified in one organism can be applied equally to otherorganisms.

The methods of the invention are applicable to various prokaryotic andeukaryotic organisms such as bacteria, yeast and fungus. For example,the yeast can include Saccharomyces cerevisiae and Rhizopus arrhizus.Exemplary eukaryotic organisms can also include Crabtree positive andnegative yeasts, and yeasts in the genera Saccharomyces, Kluyveromyces,Candida or Pichia. Further exemplary eukaryotic species include thoseselected from Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger,Rhizopus arrhizus, Rhizopus oryzae, Candida albicans, Candida boidinii,Candida sonorensis, Candida tropicalis, Yarrowia lipolytica and Pichiapastoris. Additionally, select cells from larger eukaryotic organismsare also applicable to methods of the present invention. Exemplarybacteria include species selected from Escherichia coli, Klebsiellaoxytoca, Anaerobiospirillum succiniciproducens, Actinobacillussuccinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillussubtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonasmobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomycescoelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, andPseudomonas putida.

In some aspects of the invention, production of fatty alcohols, fattyaldehydes and fatty acids through the MI-FAE cycle and terminationpathways disclosed herein are particularly useful because the cycle andpathways result in higher product and ATP yields than through naturallyoccurring biosynthetic pathways such as the well-known malonyl-CoAdependent fatty acid synthesis pathway, or in some aspects themalonyl-ACP dependent fatty acid synthesis pathway. For example, usingacetyl-CoA as a C₂ extension unit (e.g. step A, FIG. 1) instead ofmalonyl-acyl carrier protein (malonyl-ACP) saves one ATP molecule perunit flux of acetyl-CoA entering the MI-FAE cycle. The MI-FAE cycleresults in acyl-CoA instead of acyl-ACP, and can preclude the need ofthe ATP-consuming acyl-CoA synthase reactions for the production ofoctanol and other fatty alcohols, fatty aldehydes or fatty acids ifacetyl-CoA is used as the extender unit. The fatty alcohol, fattyaldehyde and fatty acid producing organisms of the invention canadditionally allow the use of biosynthetic processes to convert low costrenewable feedstock for the manufacture of chemical products.

The eukaryotic organism of the invention can be further engineered tometabolize and/or co-utilize a variety of feedstocks including glucose,xylose, fructose, syngas, methanol, and the like.

Chain length control can be achieved using a combination of highlyactive enzymes with suitable substrate ranges appropriate forbiosynthesis of the desired fatty alcohol, fatty aldehyde, or fattyacid. Chain length of the product can be controlled using one or moreenzymes of MI-FAE cycle, MD-FAE cycle or termination pathway. Asdescribed herein, chain length can be capped during the MI-FAE cycle byone or more MI-FAE cycle enzymes (thiolase, 3-oxoacyl-CoA reductase,3-hydroxyacyl-CoA dehydratase and/or enoyl-CoA reductase) and in thecase of the MD-FAE cycle, one or more MD-FAE cycle enzymes (elongase,3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and/or enoyl-CoAreductase), exhibiting selectivity for MI-FAE and/or MD-FAE cyclesubstrates having a number of carbon atoms that are no greater than thedesired product size. Since enzymes are reversible, any of theelongation pathway enzymes can serve in this capacity. Selecting enzymeswith broad substrate ranges but defined chain-length boundaries enablesthe use of a single enzyme to catalyze multiple cycles of elongation,while conferring product specificity. To further hone specificity andprevent the accumulation of shorter byproducts, selectivity is furtherconstrained by product-forming termination enzymes, such that one ormore enzymes are selective for acyl-CoA or other termination pathwaysubstrates having a number of carbon atoms that are no less than thedesired chain length. The deletion or attenuation of endogenous pathwayenzymes that produce different chain length products can further honeproduct specificity.

Using the approaches outlined herein, one skilled in the art can selectenzymes from the literature with characterized substrate ranges thatselectively produce a fatty alcohol, fatty aldehyde or fatty acidproduct of a specific chain length. To selectively produce fattyalcohols, fatty aldehydes or fatty acids of a desired length, one canutilize combinations of known enzymes in the literature with differentselectivity ranges as described above. For example, a non-naturallyoccurring microbial organism that produces C₁₆ fatty alcohol can expressenzymes such as the Rattus norvegicus Acaa1a thiolase and the enoyl-CoAreducatse of Mycobacterium smegmatis, which only accept substrates up tolength C₁₆. Coupling one or both chain elongation enzymes with a C₁₆-C₁₈fatty acyl-CoA reductase (alcohol or aldehyde forming) such as FAR ofSimmondsia chinensis further increases product specificity by reducingthe synthesis of shorter alcohol products. As another example, anon-naturally occurring microbial organism of the invention canselectively produce alcohols of length C₁₄ by combining the3-hydroxyacyl-CoA dehydratase of Arabidopsis thaliana with the acyl-CoAreductase Acr1 of Acinetobacter sp. Strain M-1. To produce 3-oxoacids oflength C₁₄, one can, for example, combine the rat thiolase with the3-oxoacyl-CoA hydrolase of Solanum lycopersicum. As still a furtherexample, to produce C₁₈ fatty acids, one can combine the Salmonellaenterica fadE enoyl-CoA reductase with the tesB thioesterase of E. coli.In yet another example, selective production of C₆ alcohols are formedby combining the paaH1 thiolase from Ralstonia eutropha with theLeifsonia sp. S749 alcohol dehydrogenase lsadh.

Exemplary MI-FAE cycle, MD-FAE cycle and termination pathway enzymes aredescribed in detail in Example I. The biosynthetic enzymes describedherein exhibit varying degrees of substrate specificity. Exemplarysubstrate ranges of enzymes characterized in the literature are shown inthe table below and described in further detail in Example I.

Pathway Chain step length Gene Organism 1A C4 atoB Escherichia coli 1AC6 phaD Pseudomonas putida 1A C6-C8 bktB Ralstonia eutropha 1A C10-C16Acaa1a Rattus norvegicus 1B C4 hbd Clostridium acetobutylicum 1B C4-C6paaH1 Ralstonia eutropha 1B C4-C10 HADH Sus scrofa 1B C4-C18 fadBEscherichia coli 1C C4-C6 crt Clostridium acetobutylicum 1C C4-C7 pimFRhodopseudomonas palustris 1C C4-C14 MFP2 Arabidopsis thaliana 1D C4-C6ECR1 Euglena gracilis 1D C6-C8 ECR3 Euglena gracilis 1D C8-10 ECR2Euglena gracilis 1D C8-C16 ECR Rattus norvegicus 1D C10-C16 ECRMycobacterium smegmatis 1D C2-C18 fadE Salmonella enterica 1E C2-C4 bphGPseudomonas sp 1E C4 Bld Clostridium saccharoperbutylacetonicum 1EC12-C20 ACR Acinetobacter calcoaceticus 1E C14-C18 Acr1 Acinetobactersp. Strain M-1 1E C16-C18 Rv1543, Mycobacterium tuberculosis Rv3391 1FC6-C7 lsadh Leifsonia sp. S749 1F C2-C8 yqhD Escherichia coli 1F C3-C10Adh Pseudomonas putida 1F C2-C14 alrA Acinetobacter sp. strain M-1 1FC2-C30 ADH1 Geobacillus thermodenitrificans 1G C2 adhE Escherichia coli1G C2-C8 adhe2 Clostridium acetobutylicum 1G C14-C16 At3g11980Arabidopsis thaliana 1G C16 At3g44560 Arabidopsis thaliana 1G C16-C18FAR Simmondsia chinensis 1H C4 Cat2 Clostridium kluyveri 1H C4-C6 Acot12Rattus norvegicus 1H C14 MKS2 Solanum lycopersicum 1L C8-C10 fatB2Cuphea hookeriana 1L C12 fatB Umbellularia california 1L C14-C16 fatB3Cuphea hookeriana 1L C18 tesA Escherichia coli 1N C12-C18 Car Nocardiaiowensis 1N C12-C16 Car Mycobacterium sp. (strain JLS) 1O C4-C8 ELO1Trypanosoma brucei 1O C10-C12 ELO2 Trypanosoma brucei 1O C14-C16 ELO3Trypanosoma brucei 1O C14-C16 ELO1 Saccharomyces cerevisiae 1O C18-C20ELO2 Saccharomyces cerevisiae 1O C22-C24 ELO3 Saccharomyces cerevisiae

Taking into account the differences in chain-length specificities ofeach enzyme in the MI-FAE cycle or MD-FAE cycle, one skilled in the artcan select one or more enzymes for catalyzing each elongation cyclereaction step (steps A-D or steps E/B/C/D of FIG. 6). For example, forthe thiolase step of the MI-FAE cycle, some thiolase enzymes such asbktB of Ralstonia eutropha catalyze the elongation of short- andmedium-chain acyl-CoA intermediates (C₆-C₈), whereas others such asAcaa1a of R. norvegicus are active on longer-chain substrates (C₁₀-C₁₆).Thus, an microbial organism producing a fatty alcohol, fatty aldehyde orfatty acid can comprise one, two, three, four or more variants of athiolase, elongase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoAdehydratase and/or enoyl-CoA reductase.

Chain length specificity of enzymes can be assayed by methods well knownin the art (eg. Wrensford et al, Anal Biochem 192:49-54 (1991)). Thesubstrate ranges of fatty alcohol, fatty aldehyde, or fatty acidproducing enzymes can be further extended or narrowed by methods wellknown in the art. Variants of biologically-occurring enzymes can begenerated, for example, by rational and directed evolution, mutagenesisand enzyme shuffling as described herein. As one example, a rationalengineering approach for altering chain length specificity was taken byDenic and Weissman (Denic and Weissman, Cell 130:663-77 (2008)). Denicand Weissman mapped the region of the yeast elongase protein ELOpresponsible for chain length, and introduced mutations to vary thelength of fatty acid products. In this instance, the geometry of thehydrophobic substrate pocket set an upper boundary on chain length. Asimilar approach can be useful for altering the chain lengthspecificities of enzymes of the MI-FAE cycle, MD-FAE cycle and/ortermination pathways.

Enzyme mutagenesis, expression in a host, and screening for fattyalcohol production is another useful approach for generating enzymevariants with improved properties for the desired application. Forexample, US patent application 2012/0009640 lists hundreds of variantsof Marinobacter algicola and Marinobacter aquaeolei FAR enzymes withimproved activity over the wild type enzyme, and varying productprofiles.

Enzyme mutagenesis (random or directed) in conjunction with a selectionplatform is another useful approach. For example, Machado and coworkersdeveloped a selection platform aimed at increasing the activity ofacyl-CoA elongation cycle enzymes on longer chain length substrates(Machado et al., Met Eng in press (2012)). Machado et al. identified thechain-length limiting step of their pathway (a 3-hydroxyacyl-CoAdehydrogenase) and evolved it for improved activity on C₆-C₈ substratesusing an anaerobic growth rescue platform. Additional variants ofenzymes useful for producing fatty alcohols are listed in the tablebelow

Protein/ GenBankID/ Enzyme GI number Organism Variant(s) Reference3-Ketoacyl- Acaa2 Rattus H352A, H352E, Zeng et al., Prot. CoANP_569117.1 norvegicus H352K, H352Y Expr. Purif. 35: thiolaseGI:18426866 320-326 (2004) 3-Hydroxyacyl- Hadh Rattus S137A, S137C, Liuet al., Prot. CoA NP_476534.1 norvegicus S137T Expr. Purif. 37:dehydrogenase GI:17105336 344-351 (2004). Enoyl-CoA Ech1 Rattus E144A,Kiema et al., hydratase NP_072116.1 norvegicus E144A/Q162L, Biochem. 38:GI:12018256 E164A, Q162A, 2991-2999 (1999) Q162L, Q162M Enoyl-CoA InhAMycobacterium K165A, K165Q, Poletto, S. et al., reductase AAY54545.1tuberculosis Y158F Prot. Expr. Purif. GI:66737267 34: 118-125 (2004).Acyl-CoA LuxC Photobacterium C171S, C279S, Lee, C. et al., reductaseAAT00788.1 phosphoreum C286S Biochim. Biophys. GI:46561111 Acta. 1338:215-222 (1997). Alcohol YADH-1 Saccharomyces D223G, D49N, Leskovac etal., dehydrogenase P00330.4 cerevisiae E68Q, G204A, FEMS Yeast Res.GI:1168350 G224I, H47R, 2(4): 481-94 (2002). H51E, L203A Fatty alcoholAdhE Escherichia A267T/E568K, Membrillo et al., forming acyl-CoANP_415757.1 coli A267T J. Biol. Chem. reductase (FAR) GI:16129202275(43): 333869-75 (2000).

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli or S. cerevisiaeand their corresponding metabolic reactions or a suitable sourceorganism for desired genetic material such as genes for a desiredmetabolic pathway. However, given the complete genome sequencing of awide variety of organisms and the high level of skill in the area ofgenomics, those skilled in the art will readily be able to apply theteachings and guidance provided herein to essentially all otherorganisms. For example, the metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having fatty alcohol, fattyaldehyde or fatty acid biosynthetic capability, those skilled in the artwill understand with applying the teaching and guidance provided hereinto a particular species that the identification of metabolicmodifications can include identification and inclusion or inactivationof orthologs. To the extent that paralogs and/or nonorthologous genedisplacements are present in the referenced microorganism that encode anenzyme catalyzing a similar or substantially similar metabolic reaction,those skilled in the art also can utilize these evolutionally relatedgenes. Similarly for a gene disruption, evolutionally related genes canalso be disrupted or deleted in a host microbial organism to reduce oreliminate functional redundancy of enzymatic activities targeted fordisruption.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a MI-FAE cycle or a MD-FAE cycle incombination with a termination pathway, wherein the MI-FAE cycleincludes one or more thiolase, one or more 3-oxoacyl-CoA reductase, oneor more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoAreductase, wherein the MD-FAE cycle includes one or more elongase, oneor more 3-oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoAdehydratase, and one or more enoyl-CoA reductase, wherein thetermination pathway includes a pathway shown in FIG. 1, 6 or 7 selectedfrom: (1) 1H; (2) 1K and 1L; (3) 1E and 1N; (4) 1K, 1J, and 1N; (5) 1E;(6) 1K and 1J; (7) 1H and 1N; (8) 1K, 1L, and 1N; (9) 1E and 1F; (10)1K, 1J, and 1F; (11) 1H, 1N, and 1F; (12) 1K, 1L, 1N, and 1F; and (13)1G, wherein 1E is an acyl-CoA reductase (aldehyde forming), wherein 1Fis an alcohol dehydrogenase, wherein 1G is an acyl-CoA reductase(alcohol forming), wherein 1H is an acyl-CoA hydrolase, acyl-CoAtransferase or acyl-CoA synthase, wherein 1J is an acyl-ACP reductase,wherein 1K is an acyl-CoA:ACP acyltransferase, wherein 1L is athioesterase, wherein 1N is an aldehyde dehydrogenase (acid forming) ora carboxylic acid reductase, wherein an enzyme of the MI-FAE cycle,MD-FAE cycle or termination pathway is encoded by at least one exogenousnucleic acid and is expressed in a sufficient amount to produce acompound of Formula (I):

wherein R₁ is C₁₋₂₄ linear alkyl; R₂ is CH₂OH, CHO, or COOH; R₃ is H,OH, or oxo (═O); and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four, wherein the substrateof each of said enzymes of the MI-FAE cycle, MD-FAE cycle and thetermination pathway are independently selected from a compound ofFormula (II), malonyl-CoA, propionyl-CoA or acetyl-CoA:

wherein R₁ is C₁₋₂₄ linear alkyl; R₃ is H, OH, or oxo (═O); R₄ is S-CoA,ACP, OH or H; and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four; wherein said one ormore enzymes of the MI-FAE cycle are each selective for a compound ofFormula (II) having a number of carbon atoms at R₁ that is no greaterthan the number of carbon atoms at R₁ of said compound of Formula (I),wherein said one or more enzymes of the MD-FAE cycle are each selectivefor a compound of Formula (II) having a number of carbon atoms at R₁that is no greater than the number of carbon atoms at R₁ of saidcompound of Formula (I), and wherein said one or more enzymes of thetermination pathway are each selective for a compound of Formula (II)having a number of carbon atoms at R₁ that is no less than the number ofcarbon atoms at R₁ of said compound of Formula (I).

In some aspects of the invention, non-naturally occurring microbialorganism of the invention can produce a compound of Formula (I) whereinR₁ is C₁₋₁₇ linear alkyl. In another aspect of the invention, the R₁ ofthe compound of Formula (I) is C₁ linear alkyl, C₂ linear alkyl, C₃linear alkyl, C₄ linear alkyl, C₅ linear alkyl, C₆ linear alkyl, C₇linear alkyl, C₈ linear alkyl, C₉ linear alkyl, C₁₀ linear alkyl, C₁₁,linear alkyl, C₁₂ linear alkyl or C₁₃ linear alkyl, C₁₄ linear alkyl,C₁₅ linear alkyl, C₁₆ linear alkyl, C₁₇ linear alkyl, C₁₈ linear alkyl,C₁₉ linear alkyl, C₂₀ linear alkyl, C₂₁ linear alkyl, C₂₂ linear alkyl,C₂₃ linear alkyl, or C₂₄ linear alkyl.

In some aspects of the invention, the microbial organism microbialorganism includes two, three, or four exogenous nucleic acids eachencoding an enzyme of the MI-FAE cycle or the MD-FAE cycle. In someaspects of the invention, the microbial organism includes two, three, orfour exogenous nucleic acids each encoding an enzyme of the terminationpathway. In some aspects of the invention, the microbial organismincludes exogenous nucleic acids encoding each of the enzymes of atleast one of the pathways selected from (1)-(13). In some aspects, theat least one exogenous nucleic acid is a heterologous nucleic acid. Insome aspects, the non-naturally occurring microbial organism is in asubstantially anaerobic culture medium.

In some embodiments, the invention provides a non naturally occurringmicrobial organism, wherein the one or more enzymes of the MI-FAE cycle,MD-FAE cycle or termination pathway is expressed in a sufficient amountto produce a fatty alcohol selected from the Formulas (III)-(VI):

wherein R₁ is C₁₋₂₄ linear alkyl, or alternatively R₁ is C₁₋₁₇ linearalkyl, or alternatively R₁ is C₉₋₁₃ linear alkyl. In some aspects of theinvention, R₁ is C₁ linear alkyl, C₂ linear alkyl, C₃ linear alkyl, C₄linear alkyl, C₅ linear alkyl, C₆ linear alkyl, C₇ linear alkyl, C₈linear alkyl, C₉ linear alkyl, C₁₀ linear alkyl, C₁₁, linear alkyl, C₁₂linear alkyl or C₁₃ linear alkyl, C₁₄ linear alkyl, C₁₅ linear alkyl,C₁₆ linear alkyl, C₁₇ linear alkyl, C₁₈ linear alkyl, C₁₉ linear alkyl,C_(m) linear alkyl, C₂₁ linear alkyl, C₂₂ linear alkyl, C₂₃ linearalkyl, or C₂₄ linear alkyl.

In some embodiments, the invention provides a non naturally occurringmicrobial organism, wherein the one or more enzymes of the MI-FAE cycle,MD-FAE cycle or termination pathway is expressed in a sufficient amountto produce a fatty aldehyde selected from the Formula (VII)-(X):

wherein R₁ is C₁₋₂₄ linear alkyl, or alternatively R₁ is C₁₋₁₇ linearalkyl, or alternatively R₁ is C₉₋₁₃ linear alkyl. In some aspects of theinvention, R₁ is C₁ linear alkyl, C₂ linear alkyl, C₃ linear alkyl, C₄linear alkyl, C₅ linear alkyl, C₆ linear alkyl, C₇ linear alkyl, C₈linear alkyl, C₉ linear alkyl, C₁₀ linear alkyl, C₁₁, linear alkyl, C₁₂linear alkyl or C₁₃ linear alkyl, C₁₄ linear alkyl, C₁₅ linear alkyl,C₁₆ linear alkyl, C₁₇ linear alkyl, C₁₈ linear alkyl, C₁₉ linear alkyl,C₂₀ linear alkyl, C₂₁ linear alkyl, C₂₂ linear alkyl, C₂₃ linear alkyl,or C₂₄ linear alkyl.

In some embodiments, the invention provides a non naturally occurringmicrobial organism, wherein the one or more enzymes of the MI-FAE cycle,MD-FAE cycle or termination pathway is expressed in a sufficient amountto produce a fatty acid selected from the Formula (XI)-(XIV):

wherein R₁ is C₁₋₂₄ linear alkyl, or alternatively R₁ is C₁₋₁₇ linearalkyl, or alternatively R₁ is C₉₋₁₃ linear alkyl. In some aspects of theinvention, R₁ is C₁ linear alkyl, C₂ linear alkyl, C₃ linear alkyl, C₄linear alkyl, C₅ linear alkyl, C₆ linear alkyl, C₇ linear alkyl, C₈linear alkyl, C₉ linear alkyl, C₁₀ linear alkyl, C₁₁, linear alkyl, C₁₂linear alkyl or C₁₃ linear alkyl, C₁₄ linear alkyl, C₁₅ linear alkyl,C₁₆ linear alkyl, C₁₇ linear alkyl, C₁₈ linear alkyl, C₁₉ linear alkyl,C₂₀ linear alkyl, C₂₁ linear alkyl, C₂₂ linear alkyl, C₂₃ linear alkyl,or C₂₄ linear alkyl.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein, wherein the microbial organismfurther includes an acetyl-CoA pathway and at least one exogenousnucleic acid encoding an acetyl-CoA pathway enzyme expressed in asufficient amount to produce acetyl-CoA, wherein the acetyl-CoA pathwayincludes a pathway shown in FIG. 2, 3, 4 or 5 selected from: (1) 2A and2B; (2) 2A, 2C, and 2D; (3) 2H; (4) 2G and 2D; (5) 2E, 2F and 2B; (6) 2Eand 2I; (7) 2J, 2F and 2B; (8) 2J and 21; (9) 3A, 3B, and 3C; (10) 3A,3B, 3J, 3K, and 3D; (11) 3A, 3B, 3G, and 3D; (12) 3A, 3F, and 3D; (13)3N, 3H, 3B and 3C; (14) 3N, 3H, 3B, 3J, 3K, and 3D; (15) 3N, 3H, 3B, 3G,and 3D; (16) 3N, 3H, 3F, and 3D; (17) 3L, 3M, 3B and 3C; (18) 3L, 3M,3B, 3J, 3K, and 3D; (19) 3L, 3M, 3B, 3G, and 3D; (20) 3L, 3M, 3F, and3D; (21) 4A, 4B, 4D, 4H, 4I, and 4J; (22) 4A, 4B, 4E, 4F, 4H, 4I, and4J; (23) 4A, 4B, 4E, 4K, 4L, 4H, 4I, and 4J; (24) 4A, 4C, 4D, 4H, and4J; (25) 4A, 4C, 4E, 4F, 4H, and 4J; (26) 4A, 4C, 4E, 4K, 4L, 4H, and4J; (27) 5A, 5B, 5D, and 5G; (28) 5A, 5B, 5E, 5F, and 5G; (29) 5A, 5B,5E, 5K, 5L, and 5G; (30) 5A, 5C, and 5D; (31) 5A, 5C, 5E, and 5F; and(32) 5A, 5C, 5E, 5K, and 5L, wherein 2A is a pyruvate oxidase(acetate-forming), wherein 2B is an acetyl-CoA synthetase, an acetyl-CoAligase or an acetyl-CoA transferase, wherein 2C is an acetate kinase,wherein 2D is a phosphotransacetylase, wherein 2E is a pyruvatedecarboxylase, wherein 2F is an acetaldehyde dehydrogenase, wherein 2Gis a pyruvate oxidase (acetyl-phosphate forming), wherein 2H is apyruvate dehydrogenase, a pyruvate:ferredoxin oxidoreductase, apyruvate:NAD(P)H oxidoreductase or a pyruvate formate lyase, wherein 2Iis an acetaldehyde dehydrogenase (acylating), wherein 2J is a threoninealdolase, wherein 3A is a phosphoenolpyruvate (PEP) carboxylase or a PEPcarboxykinase, wherein 3B is an oxaloacetate decarboxylase, wherein 3Cis a malonate semialdehyde dehydrogenase (acetylating), wherein 3D is anacetyl-CoA carboxylase or a malonyl-CoA decarboxylase, wherein 3F is anoxaloacetate dehydrogenase or an oxaloacetate oxidoreductase, wherein 3Gis a malonate semialdehyde dehydrogenase (acylating), wherein 3H is apyruvate carboxylase, wherein 3J is a malonate semialdehydedehydrogenase, wherein 3K is a malonyl-CoA synthetase or a malonyl-CoAtransferase, wherein 3L is a malic enzyme, wherein 3M is a malatedehydrogenase or a malate oxidoreductase, wherein 3N is a pyruvatekinase or a PEP phosphatase, wherein 4A is a citrate synthase, wherein4B is a citrate transporter, wherein 4C is a citrate/malate transporter,wherein 4D is an ATP citrate lyase, wherein 4E is a citrate lyase,wherein 4F is an acetyl-CoA synthetase or an acetyl-CoA transferase,wherein 4H is a cytosolic malate dehydrogenase, wherein 4I is a malatetransporter, wherein 4J is a mitochondrial malate dehydrogenase, wherein4K is an acetate kinase, wherein 4L is a phosphotransacetylase, wherein5A is a citrate synthase, wherein 5B is a citrate transporter, wherein5C is a citrate/oxaloacetate transporter, wherein 5D is an ATP citratelyase, wherein 5E is a citrate lyase, wherein 5F is an acetyl-CoAsynthetase or an acetyl-CoA transferase, wherein 5G is an oxaloacetatetransporter, wherein 5K is an acetate kinase, and wherein 5L is aphosphotransacetylase.

In some aspects, the microbial organism of the invention can includetwo, three, four, five, six, seven or eight exogenous nucleic acids eachencoding an acetyl-CoA pathway enzyme. In some aspects, the microbialorganism includes exogenous nucleic acids encoding each of theacetyl-CoA pathway enzymes of at least one of the pathways selected from(1)-(32).

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a fatty alcohol, fatty aldehyde orfatty acid pathway, wherein the non-naturally occurring microbialorganism comprises at least one exogenous nucleic acid encoding anenzyme or protein that converts a substrate to a product selected fromthe group consisting of two acetyl-CoA molecules to a 3-ketoacyl-CoA,acetyl-CoA plus propionyl-CoA to a ketoacyl-CoA, malonyl-CoA to3-ketoacyl-CoA, a 3-ketoacyl-CoA to a 3-hydroxyacyl-CoA, a3-hydroxyacyl-CoA to an enoyl-CoA, an enoyl-CoA to an acyl-CoA, anacyl-CoA plus an acetyl-CoA to a 3-ketoacyl-CoA, an acyl-CoA plusmalonyl-CoA to a 3-ketoacyl-CoA, an acyl-CoA to a fatty aldehyde, afatty aldehyde to a fatty alcohol, an acyl-CoA to a fatty alcohol, anacyl-CoA to an acyl-ACP, an acyl-ACP to a fatty acid, an acyl-CoA to afatty acid, an acyl-ACP to a fatty aldehyde, a fatty acid to a fattyaldehyde, a fatty aldehyde to a fatty acid, pyruvate to acetate, acetateto acetyl-CoA, pyruvate to acetyl-CoA, pyruvate to acetaldehyde,threonin to acetaldehyde, acetaldehyde to acetate, acetaldehyde toacetyl-CoA, pyruvate to acetyl-phosphate, acetate to acetyl-phosphate,acetyl-phosphate to acetyl-CoA, phosphoenolpyruvate (PEP) to pyruvate,pyruvate to malate, malate to oxaloacetate, pyruvate to oxaloacetate,PEP to oxaloacetate, oxaloacetate to malonate semialdehyde, oxaloacetateto malonyl-CoA, malonate semialdehyde to malonate, malonate tomalonyl-CoA, malonate semialdehyde to malonyl-CoA, malonyl-CoA toacetyl-CoA, malonate semialdehyde to acetyl-CoA, oxaloacetate plusacetyl-CoA to citrate, citrate to oxaloacetate plus acetyl-CoA, citrateto oxaloacetate plus acetate, and oxaloacetate to malate. One skilled inthe art will understand that these are merely exemplary and that any ofthe substrate-product pairs disclosed herein suitable to produce adesired product and for which an appropriate activity is available forthe conversion of the substrate to the product can be readily determinedby one skilled in the art based on the teachings herein. Thus, theinvention provides a non-naturally occurring microbial organismcontaining at least one exogenous nucleic acid encoding an enzyme orprotein, where the enzyme or protein converts the substrates andproducts of a fatty alcohol, fatty aldehyde or fatty acid pathway, suchas that shown in FIG. 1-8.

While generally described herein as a microbial organism that contains afatty alcohol, fatty aldehyde or fatty acid pathway, it is understoodthat the invention additionally provides a non-naturally occurringmicrobial organism comprising at least one exogenous nucleic acidencoding a fatty alcohol, fatty aldehyde or fatty acid pathway enzyme orprotein expressed in a sufficient amount to produce an intermediate of afatty alcohol, fatty aldehyde or fatty acid pathway. For example, asdisclosed herein, a fatty alcohol, fatty aldehyde or fatty acid pathwayis exemplified in FIGS. 1-7. Therefore, in addition to a microbialorganism containing a fatty alcohol, fatty aldehyde or fatty acidpathway that produces fatty alcohol, fatty aldehyde or fatty acid, theinvention additionally provides a non-naturally occurring microbialorganism comprising at least one exogenous nucleic acid encoding a fattyalcohol, fatty aldehyde or fatty acid pathway enzyme, where themicrobial organism produces a fatty alcohol, fatty aldehyde or fattyacid pathway intermediate, for example, a 3-ketoacyl-CoA, a3-hydroxyacyl-CoA, an enoyl-CoA, an acyl-CoA, an acyl-ACP, acetate,acetaldehyde, acetyl-phosphate, oxaloacetate, matate, malonatesemialdehyde, malonate, malonyl-CoA, acetyl-CoA, or citrate.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the Figures, including the pathwaysof FIGS. 1-7, can be utilized to generate a non-naturally occurringmicrobial organism that produces any pathway intermediate or product, asdesired. As disclosed herein, such a microbial organism that produces anintermediate can be used in combination with another microbial organismexpressing downstream pathway enzymes to produce a desired product.However, it is understood that a non-naturally occurring microbialorganism that produces a fatty alcohol, fatty aldehyde or fatty acidpathway intermediate can be utilized to produce the intermediate as adesired product.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more fatty alcohol,fatty aldehyde or fatty acid biosynthetic pathways. Depending on thehost microbial organism chosen for biosynthesis, nucleic acids for someor all of a particular fatty alcohol, fatty aldehyde or fatty acidbiosynthetic pathway can be expressed. For example, if a chosen host isdeficient in one or more enzymes or proteins for a desired biosyntheticpathway, then expressible nucleic acids for the deficient enzyme(s) orprotein(s) are introduced into the host for subsequent exogenousexpression. Alternatively, if the chosen host exhibits endogenousexpression of some pathway genes, but is deficient in others, then anencoding nucleic acid is needed for the deficient enzyme(s) orprotein(s) to achieve fatty alcohol, fatty aldehyde or fatty acidbiosynthesis. Thus, a non-naturally occurring microbial organism of theinvention can be produced by introducing exogenous enzyme or proteinactivities to obtain a desired biosynthetic pathway or a desiredbiosynthetic pathway can be obtained by introducing one or moreexogenous enzyme or protein activities that, together with one or moreendogenous enzymes or proteins, produces a desired product such as fattyalcohol, fatty aldehyde or fatty acid.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable orsuitable to fermentation processes. Exemplary bacteria include anyspecies selected from the order Enterobacteriales, familyEnterobacteriaceae, including the genera Escherichia and Klebsiella; theorder Aeromonadales, family Succinivibrionaceae, including the genusAnaerobiospirillum; the order Pasteurellales, family Pasteurellaceae,including the genera Actinobacillus and Mannheimia; the orderRhizobiales, family Bradyrhizobiaceae, including the genus Rhizobium;the order Bacillales, family Bacillaceae, including the genus Bacillus;the order Actinomycetales, families Corynebacteriaceae andStreptomycetaceae, including the genus Corynebacterium and the genusStreptomyces, respectively; order Rhodospirillales, familyAcetobacteraceae, including the genus Gluconobacter; the orderSphingomonadales, family Sphingomonadaceae, including the genusZymomonas; the order Lactobacillales, families Lactobacillaceae andStreptococcaceae, including the genus Lactobacillus and the genusLactococcus, respectively; the order Clostridiales, familyClostridiaceae, genus Clostridium; and the order Pseudomonadales, familyPseudomonadaceae, including the genus Pseudomonas. Non-limiting speciesof host bacteria include Escherichia coli, Klebsiella oxytoca,Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes,Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis,Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis,Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor,Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonasputida.

Similarly, exemplary species of yeast or fungi species include anyspecies selected from the order Saccharomycetales, familySaccaromycetaceae, including the genera Saccharomyces, Kluyveromyces andPichia; the order Saccharomycetales, family Dipodascaceae, including thegenus Yarrowia; the order Schizosaccharomycetales, familySchizosaccaromycetaceae, including the genus Schizosaccharomyces; theorder Eurotiales, family Trichocomaceae, including the genusAspergillus; and the order Mucorales, family Mucoraceae, including thegenus Rhizopus. Non-limiting species of host yeast or fungi includeSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger,Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowialipolytica, and the like. E. coli is a particularly useful host organismsince it is a well characterized microbial organism suitable for geneticengineering. Other particularly useful host organisms include yeast suchas Saccharomyces cerevisiae. It is understood that any suitablemicrobial host organism can be used to introduce metabolic and/orgenetic modifications to produce a desired product.

Depending on the fatty alcohol, fatty aldehyde or fatty acidbiosynthetic pathway constituents of a selected host microbial organism,the non-naturally occurring microbial organisms of the invention willinclude at least one exogenously expressed fatty alcohol, fatty aldehydeor fatty acid pathway-encoding nucleic acid and up to all encodingnucleic acids for one or more fatty alcohol, fatty aldehyde or fattyacid biosynthetic pathways. For example, fatty alcohol, fatty aldehydeor fatty acid biosynthesis can be established in a host deficient in apathway enzyme or protein through exogenous expression of thecorresponding encoding nucleic acid. In a host deficient in all enzymesor proteins of a fatty alcohol, fatty aldehyde or fatty acid pathway,exogenous expression of all enzyme or proteins in the pathway can beincluded, although it is understood that all enzymes or proteins of apathway can be expressed even if the host contains at least one of thepathway enzymes or proteins. For example, exogenous expression of allenzymes or proteins in a pathway for production of fatty alcohol, fattyaldehyde or fatty acid can be included, such as a thiolase, a3-oxoacyl-CoA reductase, a 3-hydroxyacyl-CoA dehydratase, an enoyl-CoAredutase, an acyl-CoA reductase (aldehyde forming) and an alcoholdehydrogenase, for production of a fatty alcohol.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the fattyalcohol, fatty aldehyde or fatty acid pathway deficiencies of theselected host microbial organism. Therefore, a non-naturally occurringmicrobial organism of the invention can have one, two, three, four,five, six, seven or eight up to all nucleic acids encoding the enzymesor proteins constituting a fatty alcohol, fatty aldehyde or fatty acidbiosynthetic pathway disclosed herein. In some embodiments, thenon-naturally occurring microbial organisms also can include othergenetic modifications that facilitate or optimize fatty alcohol, fattyaldehyde or fatty acid biosynthesis or that confer other usefulfunctions onto the host microbial organism. One such other functionalitycan include, for example, augmentation of the synthesis of one or moreof the fatty alcohol, fatty aldehyde or fatty acid pathway precursorssuch as acetyl-CoA, malonyl-CoA or propionyl-CoA.

Generally, a host microbial organism is selected such that it producesthe precursor of a fatty alcohol, fatty aldehyde or fatty acid pathway,either as a naturally produced molecule or as an engineered product thateither provides de novo production of a desired precursor or increasedproduction of a precursor naturally produced by the host microbialorganism. For example, acetyl-CoA is produced naturally in a hostorganism such as E. coli. A host organism can be engineered to increaseproduction of a precursor, as disclosed herein. In addition, a microbialorganism that has been engineered to produce a desired precursor can beused as a host organism and further engineered to express enzymes orproteins of a fatty alcohol, fatty aldehyde or fatty acid pathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize fatty alcohol, fatty aldehyde or fatty acid. Inthis specific embodiment it can be useful to increase the synthesis oraccumulation of a fatty alcohol, fatty aldehyde or fatty acid pathwayproduct to, for example, drive fatty alcohol, fatty aldehyde or fattyacid pathway reactions toward fatty alcohol, fatty aldehyde or fattyacid production. Increased synthesis or accumulation can be accomplishedby, for example, overexpression of nucleic acids encoding one or more ofthe above-described fatty alcohol, fatty aldehyde or fatty acid pathwayenzymes or proteins. Overexpression of the enzyme or enzymes and/orprotein or proteins of the fatty alcohol, fatty aldehyde or fatty acidpathway can occur, for example, through exogenous expression of theendogenous gene or genes, or through exogenous expression of theheterologous gene or genes. Therefore, naturally occurring organisms canbe readily generated to be non-naturally occurring microbial organismsof the invention, for example, producing fatty alcohol, fatty aldehydeor fatty acid, through overexpression of one, two, three, four, five,six, seven, or eight, that is, up to all nucleic acids encoding fattyalcohol, fatty aldehyde or fatty acid biosynthetic pathway enzymes orproteins. In addition, a non-naturally occurring organism can begenerated by mutagenesis of an endogenous gene that results in anincrease in activity of an enzyme in the fatty alcohol, fatty aldehydeor fatty acid biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a fatty alcohol, fatty aldehyde or fatty acid biosyntheticpathway onto the microbial organism. Alternatively, encoding nucleicacids can be introduced to produce an intermediate microbial organismhaving the biosynthetic capability to catalyze some of the requiredreactions to confer fatty alcohol, fatty aldehyde or fatty acidbiosynthetic capability. For example, a non-naturally occurringmicrobial organism having a fatty alcohol, fatty aldehyde or fatty acidbiosynthetic pathway can comprise at least two exogenous nucleic acidsencoding desired enzymes or proteins, such as the combination of athiolase and an acyl-CoA reductase (alcohol forming), or alternatively a2-oxoacyl-CoA reductase and an acyl-CoA hydrolase, or alternatively aenoyl-CoA reductase and an acyl-CoA reductase (aldehyde forming), andthe like. Thus, it is understood that any combination of two or moreenzymes or proteins of a biosynthetic pathway can be included in anon-naturally occurring microbial organism of the invention. Similarly,it is understood that any combination of three or more enzymes orproteins of a biosynthetic pathway can be included in a non-naturallyoccurring microbial organism of the invention, for example, a thiolase,an enoyl-CoA reductase and a aldehyde dehydrogenase (acid forming), oralternatively a 3-hydroxyacyl-coA dehydratase, an acyl-CoA:ACPacyltransferase and a thioesterase, or alternatively a 3-oxoacyl-CoAreductase, an acyl-CoA hydrolase and a carboxylic acid reductase, and soforth, as desired, so long as the combination of enzymes and/or proteinsof the desired biosynthetic pathway results in production of thecorresponding desired product. Similarly, any combination of four, five,six, seven, eight or more enzymes or proteins of a biosynthetic pathwayas disclosed herein can be included in a non-naturally occurringmicrobial organism of the invention, as desired, so long as thecombination of enzymes and/or proteins of the desired biosyntheticpathway results in production of the corresponding desired product.

In addition to the biosynthesis of fatty alcohol, fatty aldehyde orfatty acid as described herein, the non-naturally occurring microbialorganisms and methods of the invention also can be utilized in variouscombinations with each other and/or with other microbial organisms andmethods well known in the art to achieve product biosynthesis by otherroutes. For example, one alternative to produce fatty alcohol, fattyaldehyde or fatty acid other than use of the fatty alcohol, fattyaldehyde or fatty acid producers is through addition of anothermicrobial organism capable of converting a fatty alcohol, fatty aldehydeor fatty acid pathway intermediate to fatty alcohol, fatty aldehyde orfatty acid. One such procedure includes, for example, the fermentationof a microbial organism that produces a fatty alcohol, fatty aldehyde orfatty acid pathway intermediate. The fatty alcohol, fatty aldehyde orfatty acid pathway intermediate can then be used as a substrate for asecond microbial organism that converts the fatty alcohol, fattyaldehyde or fatty acid pathway intermediate to fatty alcohol, fattyaldehyde or fatty acid. The fatty alcohol, fatty aldehyde or fatty acidpathway intermediate can be added directly to another culture of thesecond organism or the original culture of the fatty alcohol, fattyaldehyde or fatty acid pathway intermediate producers can be depleted ofthese microbial organisms by, for example, cell separation, and thensubsequent addition of the second organism to the fermentation broth canbe utilized to produce the final product without intermediatepurification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, fatty alcohol,fatty aldehyde or fatty acid. In these embodiments, biosyntheticpathways for a desired product of the invention can be segregated intodifferent microbial organisms, and the different microbial organisms canbe co-cultured to produce the final product. In such a biosyntheticscheme, the product of one microbial organism is the substrate for asecond microbial organism until the final product is synthesized. Forexample, the biosynthesis of fatty alcohol, fatty aldehyde or fatty acidcan be accomplished by constructing a microbial organism that containsbiosynthetic pathways for conversion of one pathway intermediate toanother pathway intermediate or the product. Alternatively, fattyalcohol, fatty aldehyde or fatty acid also can be biosyntheticallyproduced from microbial organisms through co-culture or co-fermentationusing two organisms in the same vessel, where the first microbialorganism produces a fatty alcohol, fatty aldehyde or fatty acidintermediate and the second microbial organism converts the intermediateto fatty alcohol, fatty aldehyde or fatty acid.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce fatty alcohol, fattyaldehyde or fatty acid.

Similarly, it is understood by those skilled in the art that a hostorganism can be selected based on desired characteristics forintroduction of one or more gene disruptions to increase production offatty alcohol, fatty aldehyde or fatty acid. Thus, it is understoodthat, if a genetic modification is to be introduced into a host organismto disrupt a gene, any homologs, orthologs or paralogs that catalyzesimilar, yet non-identical metabolic reactions can similarly bedisrupted to ensure that a desired metabolic reaction is sufficientlydisrupted. Because certain differences exist among metabolic networksbetween different organisms, those skilled in the art will understandthat the actual genes disrupted in a given organism may differ betweenorganisms. However, given the teachings and guidance provided herein,those skilled in the art also will understand that the methods of theinvention can be applied to any suitable host microorganism to identifythe cognate metabolic alterations needed to construct an organism in aspecies of interest that will increase fatty alcohol, fatty aldehyde orfatty acid biosynthesis. In a particular embodiment, the increasedproduction couples biosynthesis of fatty alcohol, fatty aldehyde orfatty acid to growth of the organism, and can obligatorily coupleproduction of fatty alcohol, fatty aldehyde or fatty acid to growth ofthe organism if desired and as disclosed herein.

Sources of encoding nucleic acids for a fatty alcohol, fatty aldehyde orfatty acid pathway enzyme or protein can include, for example, anyspecies where the encoded gene product is capable of catalyzing thereferenced reaction. Such species include both prokaryotic andeukaryotic organisms including, but not limited to, bacteria, includingarchaea and eubacteria, and eukaryotes, including yeast, plant, insect,animal, and mammal, including human. Exemplary species for such sourcesinclude, for example, Escherichia coli, 255956237 Penicilliumchrysogenum Wisconsin 54-1255, Acetobacter pasteurians, Acidaminococcusfermentans, Acinetobacter bayliyi, Acinetobacter calcoaceticus,Acinetobacter sp. ADP1, Acinetobacter sp. Strain M-1, Actinobacillussuccinogenes, Aedes aegypti, Agrobacterium tumefaciens, Alkaliphilusmetalliredigens QYMF, Alkaliphilus oremlandii OhILAs, Anabaenavariabilis ATCC 29413, Anaerobiospirillum succiniciproducens, Anophelesgambiae str. PEST, Apis mellifera, Aquifex aeolicus, Arabidopsisthaliana, Archaeoglobus fulgidus, Archaeoglobus fulgidus DSM 4304,Ascaris suum, Aspergillus fumigatus, Aspergillus nidulans, Aspergillusniger, Aspergillus niger CBS 513.88, Aspergillus terreus NIH2624,Azotobacter vinelandii DJ, Bacillus cereus, Bacillus megaterium,Bacillus methanolicus MGA3, Bacillus methanolicus PB1, Bacillus sp.SG-1, Bacillus subtilis, Bacillus weihenstephanensis KBAB4, Bacteroidesfragilis, Bombyx mori, Bos taurus, Bradyrhizobium japonicum,Bradyrhizobium japonicum USDA110, Brassica napsus, Burkholderiaambifaria AMMD, Burkholderia multivorans ATCC 17616, Burkholderiaphymatum, Burkholderia stabilis, butyrate producing bacterium L2-50,Caenorhabditis briggsae AF16, Caenorhabditis elegans, Campylobacterjejuni, Candida albicans, Candida boidinii, Candida methylica, Candidaparapsilosis, Candida tropicalis, Candida tropicalis MYA-3404,Candidatus Protochlamydia amoebophila, Canis lupus familiaris (dog),Carboxydothermus hydrogenoformans, Carthamus tinctorius, Chlamydomonasreinhardtii, Chlorobium limicola, Chlorobium tepidum, Chloroflexusaurantiacus, Citrus junos, Clostridium acetobutylicum, Clostridiumaminobutyricum, Clostridium beijerinckii, Clostridium beijerinckii NCIMB8052, Clostridium carboxidivorans P7, Clostridium kluyveri, Clostridiumkluyveri DSM 555, Clostridium pasteurianum, Clostridiumsaccharoperbutylacetonicum, Clostridium symbiosum, Clostridium tetaniE88, Colwellia psychrerythraea 34H, Corynebacterium glutamicum,Cryptococcus neoformans var, Cryptosporidium parvum Iowa II, Cupheahookeriana, Cuphea palustris, Cupriavidus necator, Cupriavidustaiwanensis, Cyanobium PCC7001, Cyanothece sp. PCC 7425, Danio rerio,Desulfatibacillum alkenivorans AK-01, Desulfococcus oleovorans Hxd3,Desulfovibrio africanus, Dictyostelium discoideum, Dictyosteliumdiscoideum AX4, Drosophila melanogaster, Erythrobacter sp. NAP1,Escherichia coli K-12 MG1655, Euglena gracilis, Flavobacteria bacteriumBAL38, Fusobacterium nucleatum, Geobacillus thermodenitrificans,Haemophilus influenza, Haloarcula marismortui, Haloarcula marismortuiATCC 43049, Halomonas sp. HTNK1, Helianthus annuus, Helicobacter pylori,Helicobacter pylori 26695, Homo sapiens, Hydrogenobacter thermophilus,Klebsiella pneumoniae, Kluyveromyces lactis, Kluyveromyces lactis NRRLY-1140, Lactobacillus casei, Lactobacillus plantarum, Lactobacillusreuteri, Lactococcus lactis, Leifsonia sp. S749, Leuconostocmesenteroides, Lyngbya sp. PCC 8106, Macaca mulatta, Magnetospirillummagneticum AMB-1, Mannheimia succiniciproducens, marine gammaproteobacterium HTCC2080, Marinobacter aquaeolei, Marinobacter aquaeoleiVT8, Megathyrsus maximus, Mesorhizobium loti, Metallosphaera sedula,Methanosarcina thermophile, Methanothermobacter thermautotrophicus,Methylobacterium extorquens, Monosiga brevicollis MX1, Moorellathermoacetica, Moorella thermoacetica ATCC 39073, Mus musculus,Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium bovisBCG, Mycobacterium marinum M, Mycobacterium smegmatis, Mycobacteriumsmegmatis MC2 155, Mycobacterium sp. (strain JLS), Mycobacterium sp.MCS, Mycobacterium sp. strain JLS, Mycobacterium tuberculosis,Myxococcus xanthus DK 1622, Nematostella vectensis, Neurospora crassaOR74A, Nicotiana tabacum, Nocardia brasiliensis, Nocardia farcinica IFM10152, Nocardia iowensis, Nodularia spumigena CCY9414, Nostoc azollae,Nostoc sp. PCC 7120, Opitutaceae bacterium TAV2, Paracoccusdenitrificans, Penicillium chrysogenum, Perkinsus marinus ATCC 50983,Photobacterium phosphoreum, Photobacterium sp. SKA34, Picea sitchensis,Pichia pastoris, Pichia pastoris GS115, Plasmodium falciparum,Porphyromonas gingivalis, Porphyromonas gingivalis W83, Prochlorococcusmarinus MIT 9312, Propionigenium modestum, Pseudomonas aeruginosa,Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens, Pseudomonasfluorescens Pf0-1, Pseudomonas knackmussii, Pseudomonas knackmussii(B13), Pseudomonas putida, Pseudomonas putida GB-1, Pseudomonas sp,Pseudomonas sp. CF600, Pseudomonas stutzeri, Pseudomonas stutzeri A1501,Pseudomonas syringae, Pyrobaculum aerophilum str. IM2, Ralstoniaeutropha, Ralstonia metallidurans, Rattus norvegicus, Reinekea sp.MED297, Rhizobium etli CFN 42, Rhizobium leguminosarum, Rhodobactersphaeroides, Rhodococcus erythropolis, Rhodococcus sp., Rhodopseudomonaspalustris, Roseiflexus castenholzii, Roseovarius sp. HTCC2601,Saccharomyces cerevisiae, Saccharomyces cerevisiae s288c, Salmonellaenteric, Salmonella enterica subsp. enterica serovar Typhimurium str.LT2, Salmonella typhimurium, Salmonella typhimurium LT2, Scheffersomycesstipitis, Schizosaccharomyces pombe, Shigella dysenteriae, Shigellasonnei, Simmondsia chinensis, Solanum lycopersicum, Sordaria macrospora,Staphylococcus aureus, Stenotrophomonas maltophilia, Streptococcusmutans, Streptococcus pneumoniae, Streptococcus sanguinis, Streptomycesanulatus, Streptomyces avermitillis, Streptomyces cinnamonensis,Streptomyces coelicolor, Streptomyces griseus subsp. griseus NBRC 13350,Streptomyces luridus, Streptomyces sp CL190, Streptomyces sp. KO-3988,Streptomyces viridochromogenes, Streptomyces wedmorensis,Strongylocentrotus purpuratus, Sulfolobus acidocaldarius, Sulfolobussolfataricus, Sulfolobus tokodaii, Sulfurihydrogenibium subterraneum,Sulfurimonas denitrificans, Sus scrofa, Synechococcus elongatus PCC6301, Synechococcus elongatus PCC 7942, Synechococcus sp. PCC 7002,Syntrophobacter fumaroxidans, Syntrophus aciditrophicus, Tetraodonnigroviridis, Thermoanaerobacter ethanolicus JW 200, Thermoanaerobacterpseudethanolicus ATCC 33223, Thermococcus litoralis, Thermoproteusneutrophilus, Thermotoga maritime, Treponema denticola, Triboliumcastaneum, Trichomonas vaginalis G3, Triticum aestivum, Trypanosomabrucei, Trypanosoma cruzi strain CL Brener, Tsukamurella paurometabolaDSM 20162, Umbellularia California, Veillonella parvula, Vibrio choleraeV51, Xenopus tropicalis, Yarrowia lipolytica, Zea mays, Zoogloearamiger, Zymomonas mobilis, Zymomonas mobilis subsp. mobilis ZM4, aswell as other exemplary species disclosed herein or available as sourceorganisms for corresponding genes. However, with the complete genomesequence available for now more than 550 species (with more than half ofthese available on public databases such as the NCBI), including 395microorganism genomes and a variety of yeast, fungi, plant, andmammalian genomes, the identification of genes encoding the requisitefatty alcohol, fatty aldehyde or fatty acid biosynthetic activity forone or more genes in related or distant species, including for example,homologues, orthologs, paralogs and nonorthologous gene displacements ofknown genes, and the interchange of genetic alterations betweenorganisms is routine and well known in the art. Accordingly, themetabolic alterations allowing biosynthesis of fatty alcohol, fattyaldehyde or fatty acid described herein with reference to a particularorganism such as E. coli can be readily applied to other microorganisms,including prokaryotic and eukaryotic organisms alike. Given theteachings and guidance provided herein, those skilled in the art willknow that a metabolic alteration exemplified in one organism can beapplied equally to other organisms.

In some instances, such as when an alternative fatty alcohol, fattyaldehyde or fatty acid biosynthetic pathway exists in an unrelatedspecies, fatty alcohol, fatty aldehyde or fatty acid biosynthesis can beconferred onto the host species by, for example, exogenous expression ofa paralog or paralogs from the unrelated species that catalyzes asimilar, yet non-identical metabolic reaction to replace the referencedreaction. Because certain differences among metabolic networks existbetween different organisms, those skilled in the art will understandthat the actual gene usage between different organisms may differ.However, given the teachings and guidance provided herein, those skilledin the art also will understand that the teachings and methods of theinvention can be applied to all microbial organisms using the cognatemetabolic alterations to those exemplified herein to construct amicrobial organism in a species of interest that will synthesize fattyalcohol, fatty aldehyde or fatty acid. A nucleic acid molecule encodinga fatty alcohol, fatty aldehyde or fatty acid pathway enzyme or proteinof the invention can also include a nucleic acid molecule thathybridizes to a nucleic acid disclosed herein by SEQ ID NO, GenBankand/or GI number or a nucleic acid molecule that hybridizes to a nucleicacid molecule that encodes an amino acid sequence disclosed herein bySEQ ID NO, GenBank and/or GI number. Hybridization conditions caninclude highly stringent, moderately stringent, or low stringencyhybridization conditions that are well known to one of skill in the artsuch as those described herein. Similarly, a nucleic acid molecule thatcan be used in the invention can be described as having a certainpercent sequence identity to a nucleic acid disclosed herein by SEQ IDNO, GenBank and/or GI number or a nucleic acid molecule that hybridizesto a nucleic acid molecule that encodes an amino acid sequence disclosedherein by SEQ ID NO, GenBank and/or GI number. For example, the nucleicacid molecule can have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a nucleic aciddescribed herein.

Stringent hybridization refers to conditions under which hybridizedpolynucleotides are stable. As known to those of skill in the art, thestability of hybridized polynucleotides is reflected in the meltingtemperature (T_(m)) of the hybrids. In general, the stability ofhybridized polynucleotides is a function of the salt concentration, forexample, the sodium ion concentration and temperature. A hybridizationreaction can be performed under conditions of lower stringency, followedby washes of varying, but higher, stringency. Reference to hybridizationstringency relates to such washing conditions. Highly stringenthybridization includes conditions that permit hybridization of onlythose nucleic acid sequences that form stable hybridized polynucleotidesin 0.018M NaCl at 65° C., for example, if a hybrid is not stable in0.018M NaCl at 65° C., it will not be stable under high stringencyconditions, as contemplated herein. High stringency conditions can beprovided, for example, by hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Hybridization conditions other than highlystringent hybridization conditions can also be used to describe thenucleic acid sequences disclosed herein. For example, the phrasemoderately stringent hybridization refers to conditions equivalent tohybridization in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. Thephrase low stringency hybridization refers to conditions equivalent tohybridization in 10% formamide, 5×Denhart's solution, 6×SSPE, 0.2% SDSat 22° C., followed by washing in 1×SSPE, 0.2% SDS, at 37° C. Denhart'ssolution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serumalbumin (BSA). 20×SSPE (sodium chloride, sodium phosphate, ethylenediamide tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2Msodium phosphate, and 0.025 M (EDTA). Other suitable low, moderate andhigh stringency hybridization buffers and conditions are well known tothose of skill in the art and are described, for example, in Sambrook etal., Molecular Cloning: A Laboratory Manual, Third Ed., Cold SpringHarbor Laboratory, New York (2001); and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1999).

A nucleic acid molecule encoding a fatty alcohol, fatty aldehyde orfatty acid pathway enzyme or protein of the invention can have at leasta certain sequence identity to a nucleotide sequence disclosed herein.According, in some aspects of the invention, a nucleic acid moleculeencoding a fatty alcohol, fatty aldehyde or fatty acid pathway enzyme orprotein has a nucleotide sequence of at least 65% identity, at least 70%identity, at least 75% identity, at least 80% identity, at least 85%identity, at least 90% identity, at least 91% identity, at least 92%identity, at least 93% identity, at least 94% identity, at least 95%identity, at least 96% identity, at least 97% identity, at least 98%identity, or at least 99% identity to a nucleic acid disclosed herein bySEQ ID NO, GenBank and/or GI number or a nucleic acid molecule thathybridizes to a nucleic acid molecule that encodes an amino acidsequence disclosed herein by SEQ ID NO, GenBank and/or GI number.

Sequence identity (also known as homology or similarity) refers tosequence similarity between two nucleic acid molecules or between twopolypeptides. Identity can be determined by comparing a position in eachsequence, which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same base or aminoacid, then the molecules are identical at that position. A degree ofidentity between sequences is a function of the number of matching orhomologous positions shared by the sequences. The alignment of twosequences to determine their percent sequence identity can be done usingsoftware programs known in the art, such as, for example, thosedescribed in Ausubel et al., Current Protocols in Molecular Biology,John Wiley and Sons, Baltimore, Md. (1999). Preferably, defaultparameters are used for the alignment. One alignment program well knownin the art that can be used is BLAST set to default parameters. Inparticular, programs are BLASTN and BLASTP, using the following defaultparameters: Genetic code=standard; filter=none; strand=both; cutoff=60;expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGHSCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the National Center for Biotechnology Information.

Methods for constructing and testing the expression levels of anon-naturally occurring fatty alcohol, fatty aldehyde or fattyacid-producing host can be performed, for example, by recombinant anddetection methods well known in the art. Such methods can be founddescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York(2001); and Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production offatty alcohol, fatty aldehyde or fatty acid can be introduced stably ortransiently into a host cell using techniques well known in the artincluding, but not limited to, conjugation, electroporation, chemicaltransformation, transduction, transfection, and ultrasoundtransformation. For exogenous expression in E. coli or other prokaryoticcells, some nucleic acid sequences in the genes or cDNAs of eukaryoticnucleic acids can encode targeting signals such as an N-terminalmitochondrial or other targeting signal, which can be removed beforetransformation into prokaryotic host cells, if desired. For example,removal of a mitochondrial leader sequence led to increased expressionin E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)).For exogenous expression in yeast or other eukaryotic cells, genes canbe expressed in the cytosol without the addition of leader sequence, orcan be targeted to mitochondrion or other organelles, or targeted forsecretion, by the addition of a suitable targeting sequence such as amitochondrial targeting or secretion signal suitable for the host cells.Thus, it is understood that appropriate modifications to a nucleic acidsequence to remove or include a targeting sequence can be incorporatedinto an exogenous nucleic acid sequence to impart desirable properties.Furthermore, genes can be subjected to codon optimization withtechniques well known in the art to achieve optimized expression of theproteins.

An expression vector or vectors can be constructed to include one ormore fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathwayencoding nucleic acids as exemplified herein operably linked toexpression control sequences functional in the host organism. Expressionvectors applicable for use in the microbial host organisms of theinvention include, for example, plasmids, phage vectors, viral vectors,episomes and artificial chromosomes, including vectors and selectionsequences or markers operable for stable integration into a hostchromosome. Additionally, the expression vectors can include one or moreselectable marker genes and appropriate expression control sequences.Selectable marker genes also can be included that, for example, provideresistance to antibiotics or toxins, complement auxotrophicdeficiencies, or supply critical nutrients not in the culture media.Expression control sequences can include constitutive and induciblepromoters, transcription enhancers, transcription terminators, and thelike which are well known in the art. When two or more exogenousencoding nucleic acids are to be co-expressed, both nucleic acids can beinserted, for example, into a single expression vector or in separateexpression vectors. For single vector expression, the encoding nucleicacids can be operationally linked to one common expression controlsequence or linked to different expression control sequences, such asone inducible promoter and one constitutive promoter. The transformationof exogenous nucleic acid sequences involved in a metabolic or syntheticpathway can be confirmed using methods well known in the art. Suchmethods include, for example, nucleic acid analysis such as Northernblots or polymerase chain reaction (PCR) amplification of mRNA, orimmunoblotting for expression of gene products, or other suitableanalytical methods to test the expression of an introduced nucleic acidsequence or its corresponding gene product. It is understood by thoseskilled in the art that the exogenous nucleic acid is expressed in asufficient amount to produce the desired product, and it is furtherunderstood that expression levels can be optimized to obtain sufficientexpression using methods well known in the art and as disclosed herein.

In some embodiments, the invention provides a method for producing acompound of Formula (I):

wherein R₁ is C₁₋₂₄ linear alkyl; R₂ is CH₂OH, CHO, or COOH; R₃ is H,OH, or oxo (═O); and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four, comprising culturing anon-naturally occurring microbial organism of under conditions and for asufficient period of time to produce the compound of Formula (I),wherein the non-naturally occurring microbial organism has a MI-FAEcycle and/or a MD-FAE cycle in combination with a termination pathway,wherein the MI-FAE cycle includes one or more thiolase, one or more3-oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, andone or more enoyl-CoA reductase, wherein the MD-FAE cycle includes oneor more elongase, one or more 3-oxoacyl-CoA reductase, one or more3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase,wherein the termination pathway includes a pathway shown in FIG. 1, 6 or7 selected from: (1) 1H; (2) 1K and 1L; (3) 1E and 1N; (4) 1K, 1J, and1N; (5) 1E; (6) 1K and 1J; (7) 1H and 1N; (8) 1K, 1L, and 1N; (9) 1E and1F; (10) 1K, 1J, and 1F; (11) 1H, 1N, and 1F; (12) 1K, 1L, 1N, and 1F;and (13) 1G, wherein 1E is an acyl-CoA reductase (aldehyde forming),wherein 1F is an alcohol dehydrogenase, wherein 1G is an acyl-CoAreductase (alcohol forming), wherein 1H is an acyl-CoA hydrolase,acyl-CoA transferase or acyl-CoA synthase, wherein 1J is an acyl-ACPreductase, wherein 1K is an acyl-CoA:ACP acyltransferase, wherein 1L isa thioesterase, wherein 1N is an aldehyde dehydrogenase (acid forming)or a carboxylic acid reductase, wherein an enzyme of the MI-FAE cycle,MD-FAE cycle or termination pathway is encoded by at least one exogenousnucleic acid and is expressed in a sufficient amount to produce thecompound of Formula (I), wherein the substrate of each of said enzymesof the MI-FAE cycle, MD-FAE cycle and the termination pathway areindependently selected from a compound of Formula (II), malonyl-CoA,propionyl-CoA or acetyl-CoA:

wherein R₁ is C₁₋₂₄ linear alkyl; R₃ is H, OH, or oxo (═O); R₄ is S-CoA,ACP, OH or H; and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four; wherein said one ormore enzymes of the MI-FAE cycle are each selective for a compound ofFormula (II) having a number of carbon atoms at R₁ that is no greaterthan the number of carbon atoms at R₁ of said compound of Formula (I),wherein said one or more enzymes of the MD-FAE cycle are each selectivefor a compound of Formula (II) having a number of carbon atoms at R₁that is no greater than the number of carbon atoms at R₁ of saidcompound of Formula (I), and wherein said one or more enzymes of thetermination pathway are each selective for a compound of Formula (II)having a number of carbon atoms at R₁ that is no less than the number ofcarbon atoms at R₁ of said compound of Formula (I).

In some embodiments, the invention provides a method for producing acompound of Formula (I) wherein R₁ is C₁₋₁₇ linear alkyl. In anotheraspect of the invention, the R₁ of the compound of Formula (I) is C₁linear alkyl, C₂ linear alkyl, C₃ linear alkyl, C₄ linear alkyl, C₅linear alkyl, C₆ linear alkyl, C₇ linear alkyl, C₈ linear alkyl, C₉linear alkyl, C₁₀ linear alkyl, C₁₁, linear alkyl, C₁₂ linear alkyl orC₁₃ linear alkyl, C₁₄ linear alkyl, C₁₅ linear alkyl, C₁₆ linear alkyl,C₁₇ linear alkyl, C₁₈ linear alkyl, C₁₉ linear alkyl, C₂₀ linear alkyl,C₂₁ linear alkyl, C₂₂ linear alkyl, C₂₃ linear alkyl, or C₂₄ linearalkyl.

In some aspects of the invention, the microbial organism microbialorganism used in the method of the invention includes two, three, orfour exogenous nucleic acids each encoding an enzyme of the MI-FAE cycleor the MD-FAE cycle. In some aspects of the invention, the microbialorganism used in the method of the invention includes two, three, orfour exogenous nucleic acids each encoding an enzyme of the terminationpathway. In some aspects of the invention, the microbial organism usedin the method of the invention includes exogenous nucleic acids encodingeach of the enzymes of at least one of the pathways selected from(1)-(13). In some aspects, the at least one exogenous nucleic acid is aheterologous nucleic acid. In some aspects, the non-naturally occurringmicrobial organism used in the method of the invention is in asubstantially anaerobic culture medium.

In some embodiments, the invention provides a method for producing afatty alcohol selected from the Formulas (III)-(VI):

wherein R₁ is C₁₋₂₄ linear alkyl, or alternatively R₁ is C₁₋₁₇ linearalkyl, or alternatively R₁ is C₉₋₁₃ linear alkyl. In some aspects of theinvention, R₁ is C₁ linear alkyl, C₂ linear alkyl, C₃ linear alkyl, C₄linear alkyl, C₅ linear alkyl, C₆ linear alkyl, C₇ linear alkyl, C₈linear alkyl, C₉ linear alkyl, C₁₀ linear alkyl, C₁₁, linear alkyl, C₁₂linear alkyl or C₁₃ linear alkyl, C₁₄ linear alkyl, C₁₅ linear alkyl,C₁₆ linear alkyl, C₁₇ linear alkyl, C₁₈ linear alkyl, C₁₉ linear alkyl,C₂₀ linear alkyl, C₂₁ linear alkyl, C₂₂ linear alkyl, C₂₃ linear alkyl,or C₂₄ linear alkyl.

In some embodiments, the invention provides a method for producing afatty aldehyde selected from the Formulas (VII)-(X):

wherein R₁ is C₁₋₂₄ linear alkyl, or alternatively R₁ is C₁₋₁₇ linearalkyl, or alternatively R₁ is C₉₋₁₃ linear alkyl. In some aspects of theinvention, R₁ is C₁ linear alkyl, C₂ linear alkyl, C₃ linear alkyl, C₄linear alkyl, C₅ linear alkyl, C₆ linear alkyl, C₇ linear alkyl, C₈linear alkyl, C₉ linear alkyl, C₁₀ linear alkyl, C₁₁, linear alkyl, C₁₂linear alkyl or C₁₃ linear alkyl, C₁₄ linear alkyl, C₁₅ linear alkyl,C₁₆ linear alkyl, C₁₇ linear alkyl, C₁₈ linear alkyl, C₁₉ linear alkyl,C₂₀ linear alkyl, C₂₁ linear alkyl, C₂₂ linear alkyl, C₂₃ linear alkyl,or C₂₄ linear alkyl.

In some embodiments, the invention provides a method for producing afatty acid selected from the Formulas (XI)-(XIV):

wherein R₁ is C₁₋₂₄ linear alkyl, or alternatively R₁ is C₁₋₁₇ linearalkyl, or alternatively R₁ is C₉₋₁₃ linear alkyl. In some aspects of theinvention, R₁ is C₁ linear alkyl, C₂ linear alkyl, C₃ linear alkyl, C₄linear alkyl, C₅ linear alkyl, C₆ linear alkyl, C₇ linear alkyl, C₈linear alkyl, C₉ linear alkyl, C₁₀ linear alkyl, C₁₁, linear alkyl, C₁₂linear alkyl or C₁₃ linear alkyl, C₁₄ linear alkyl, C₁₅ linear alkyl,C₁₆ linear alkyl, C₁₇ linear alkyl, C₁₈ linear alkyl, C₁₉ linear alkyl,C₂₀ linear alkyl, C₂₁ linear alkyl, C₂₂ linear alkyl, C₂₃ linear alkyl,or C₂₄ linear alkyl.

In some embodiments, the method for producing a fatty alcohol, fattyaldehyde or fatty acid described herein includes using a non-naturallyoccurring microbial organism that has an acetyl-CoA pathway and at leastone exogenous nucleic acid encoding an acetyl-CoA pathway enzymeexpressed in a sufficient amount to produce acetyl-CoA, wherein theacetyl-CoA pathway includes a pathway shown in FIG. 2, 3, 4 or 5selected from: (1) 2A and 2B; (2) 2A, 2C, and 2D; (3) 2H; (4) 2G and 2D;(5) 2E, 2F and 2B; (6) 2E and 2I; (7) 2J, 2F and 2B; (8) 2J and 2I; (9)3A, 3B, and 3C; (10) 3A, 3B, 3J, 3K, and 3D; (11) 3A, 3B, 3G, and 3D;(12) 3A, 3F, and 3D; (13) 3N, 3H, 3B and 3C; (14) 3N, 3H, 3B, 3J, 3K,and 3D; (15) 3N, 3H, 3B, 3G, and 3D; (16) 3N, 3H, 3F, and 3D; (17) 3L,3M, 3B and 3C; (18) 3L, 3M, 3B, 3J, 3K, and 3D; (19) 3L, 3M, 3B, 3G, and3D; (20) 3L, 3M, 3F, and 3D; (21) 4A, 4B, 4D, 4H, 4I, and 4J; (22) 4A,4B, 4E, 4F, 4H, 4I, and 4J; (23) 4A, 4B, 4E, 4K, 4L, 4H, 4I, and 4J;(24) 4A, 4C, 4D, 4H, and 4J; (25) 4A, 4C, 4E, 4F, 4H, and 4J; (26) 4A,4C, 4E, 4K, 4L, 4H, and 4J; (27) 5A, 5B, 5D, and 5G; (28) 5A, 5B, 5E,5F, and 5G; (29) 5A, 5B, 5E, 5K, 5L, and 5G; (30) 5A, 5C, and 5D; (31)5A, 5C, 5E, and 5F; and (32) 5A, 5C, 5E, 5K, and 5L, wherein 2A is apyruvate oxidase (acetate-forming), wherein 2B is an acetyl-CoAsynthetase, an acetyl-CoA ligase or an acetyl-CoA transferase, wherein2C is an acetate kinase, wherein 2D is a phosphotransacetylase, wherein2E is a pyruvate decarboxylase, wherein 2F is an acetaldehydedehydrogenase, wherein 2G is a pyruvate oxidase (acetyl-phosphateforming), wherein 2H is a pyruvate dehydrogenase, a pyruvate:ferredoxinoxidoreductase, a pyruvate:NAD(P)H oxidoreductase or a pyruvate formatelyase, wherein 2I is an acetaldehyde dehydrogenase (acylating), wherein2J is a threonine aldolase, wherein 3A is a phosphoenolpyruvate (PEP)carboxylase or a PEP carboxykinase, wherein 3B is an oxaloacetatedecarboxylase, wherein 3C is a malonate semialdehyde dehydrogenase(acetylating), wherein 3D is an acetyl-CoA carboxylase or a malonyl-CoAdecarboxylase, wherein 3F is an oxaloacetate dehydrogenase or anoxaloacetate oxidoreductase, wherein 3G is a malonate semialdehydedehydrogenase (acylating), wherein 3H is a pyruvate carboxylase, wherein3J is a malonate semialdehyde dehydrogenase, wherein 3K is a malonyl-CoAsynthetase or a malonyl-CoA transferase, wherein 3L is a malic enzyme,wherein 3M is a malate dehydrogenase or a malate oxidoreductase, wherein3N is a pyruvate kinase or a PEP phosphatase, wherein 4A is a citratesynthase, wherein 4B is a citrate transporter, wherein 4C is acitrate/malate transporter, wherein 4D is an ATP citrate lyase, wherein4E is a citrate lyase, wherein 4F is an acetyl-CoA synthetase or anacetyl-CoA transferase, wherein 4H is a cytosolic malate dehydrogenase,wherein 4I is a malate transporter, wherein 4J is a mitochondrial malatedehydrogenase, wherein 4K is an acetate kinase, wherein 4L is aphosphotransacetylase, wherein 5A is a citrate synthase, wherein 5B is acitrate transporter, wherein 5C is a citrate/oxaloacetate transporter,wherein 5D is an ATP citrate lyase, wherein 5E is a citrate lyase,wherein 5F is an acetyl-CoA synthetase or an acetyl-CoA transferase,wherein 5G is an oxaloacetate transporter, wherein 5K is an acetatekinase, and wherein 5L is a phosphotransacetylase.

In some aspects, the microbial organism used in the method of theinvention includes two, three, four, five, six, seven or eight exogenousnucleic acids each encoding an acetyl-CoA pathway enzyme. In someaspects, the microbial organism used in the method of the inventionincludes exogenous nucleic acids encoding each of the acetyl-CoA pathwayenzymes of at least one of the pathways selected from (1)-(32).

Suitable purification and/or assays to test for the production of fattyalcohol, fatty aldehyde or fatty acid can be performed using well knownmethods. Suitable replicates such as triplicate cultures can be grownfor each engineered strain to be tested. For example, product andbyproduct formation in the engineered production host can be monitored.The final product and intermediates, and other organic compounds, can beanalyzed by methods such as HPLC (High Performance LiquidChromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS(Liquid Chromatography-Mass Spectroscopy) or other suitable analyticalmethods using routine procedures well known in the art. The release ofproduct in the fermentation broth can also be tested with the culturesupernatant. Byproducts and residual glucose can be quantified by HPLCusing, for example, a refractive index detector for glucose andalcohols, and a UV detector for organic acids (Lin et al., Biotechnol.Bioeng. 90:775-779 (2005)), or other suitable assay and detectionmethods well known in the art. The individual enzyme or proteinactivities from the exogenous DNA sequences can also be assayed usingmethods well known in the art.

The fatty alcohol, fatty aldehyde or fatty acid can be separated fromother components in the culture using a variety of methods well known inthe art. Such separation methods include, for example, extractionprocedures as well as methods that include continuous liquid-liquidextraction, pervaporation, membrane filtration, membrane separation,reverse osmosis, electrodialysis, distillation, crystallization,centrifugation, extractive filtration, ion exchange chromatography, sizeexclusion chromatography, adsorption chromatography, andultrafiltration. All of the above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the fatty alcohol, fatty aldehyde or fattyacid producers can be cultured for the biosynthetic production of fattyalcohol, fatty aldehyde or fatty acid. Accordingly, in some embodiments,the invention provides culture medium having the fatty alcohol, fattyaldehyde or fatty acid pathway intermediate described herein. In someaspects, the culture medium can also be separated from the non-naturallyoccurring microbial organisms of the invention that produced the fattyalcohol, fatty aldehyde or fatty acid pathway intermediate. Methods forseparating a microbial organism from culture medium are well known inthe art. Exemplary methods include filtration, flocculation,precipitation, centrifugation, sedimentation, and the like.

For the production of fatty alcohol, fatty aldehyde or fatty acid, therecombinant strains are cultured in a medium with carbon source andother essential nutrients. It is sometimes desirable and can be highlydesirable to maintain anaerobic conditions in the fermenter to reducethe cost of the overall process. Such conditions can be obtained, forexample, by first sparging the medium with nitrogen and then sealing theflasks with a septum and crimp-cap. For strains where growth is notobserved anaerobically, microaerobic or substantially anaerobicconditions can be applied by perforating the septum with a small holefor limited aeration. Exemplary anaerobic conditions have been describedpreviously and are well-known in the art. Exemplary aerobic andanaerobic conditions are described, for example, in United Statepublication 2009/0047719, filed Aug. 10, 2007. Fermentations can beperformed in a batch, fed-batch or continuous manner, as disclosedherein. Fermentations can also be conducted in two phases, if desired.The first phase can be aerobic to allow for high growth and thereforehigh productivity, followed by an anaerobic phase of high fatty alcohol,fatty aldehyde or fatty acid yields.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example: sugars such asglucose, xylose, arabinose, galactose, mannose, fructose, sucrose andstarch; or glycerol, and it is understood that a carbon source can beused alone as the sole source of carbon or in combination with othercarbon sources described herein or known in the art. Other sources ofcarbohydrate include, for example, renewable feedstocks and biomass.Exemplary types of biomasses that can be used as feedstocks in themethods of the invention include cellulosic biomass, hemicellulosicbiomass and lignin feedstocks or portions of feedstocks. Such biomassfeedstocks contain, for example, carbohydrate substrates useful ascarbon sources such as glucose, xylose, arabinose, galactose, mannose,fructose and starch. Given the teachings and guidance provided herein,those skilled in the art will understand that renewable feedstocks andbiomass other than those exemplified above also can be used forculturing the microbial organisms of the invention for the production offatty alcohol, fatty aldehyde or fatty acid.

In addition to renewable feedstocks such as those exemplified above, thefatty alcohol, fatty aldehyde or fatty acid microbial organisms of theinvention also can be modified for growth on syngas as its source ofcarbon. In this specific embodiment, one or more proteins or enzymes areexpressed in the fatty alcohol, fatty aldehyde or fatty acid producingorganisms to provide a metabolic pathway for utilization of syngas orother gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:

2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATP

Hence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes or proteins: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes or proteins: methyltetrahydrofolate:corrinoidprotein methyltransferase (for example, AcsE), corrinoid iron-sulfurprotein, nickel-protein assembly protein (for example, AcsF),ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase andnickel-protein assembly protein (for example, CooC). Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate a fatty alcohol, fattyaldehyde or fatty acid pathway, those skilled in the art will understandthat the same engineering design also can be performed with respect tointroducing at least the nucleic acids encoding the Wood-Ljungdahlenzymes or proteins absent in the host organism. Therefore, introductionof one or more encoding nucleic acids into the microbial organisms ofthe invention such that the modified organism contains the completeWood-Ljungdahl pathway will confer syngas utilization ability.

Additionally, the reductive (reverse) tricarboxylic acid cycle coupledwith carbon monoxide dehydrogenase and/or hydrogenase activities canalso be used for the conversion of CO, CO₂ and/or H₂ to acetyl-CoA andother products such as acetate. Organisms capable of fixing carbon viathe reductive TCA pathway can utilize one or more of the followingenzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitratedehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase,succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase,fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase,carbon monoxide dehydrogenase, and hydrogenase. Specifically, thereducing equivalents extracted from CO and/or H₂ by carbon monoxidedehydrogenase and hydrogenase are utilized to fix CO₂ via the reductiveTCA cycle into acetyl-CoA or acetate. Acetate can be converted toacetyl-CoA by enzymes such as acetyl-CoA transferase, acetatekinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA canbe converted to the fatty alcohol, fatty aldehyde or fatty acidprecursors, glyceraldehyde-3-phosphate, phosphoenolpyruvate, andpyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes ofgluconeogenesis. Following the teachings and guidance provided hereinfor introducing a sufficient number of encoding nucleic acids togenerate a fatty alcohol, fatty aldehyde or fatty acid pathway, thoseskilled in the art will understand that the same engineering design alsocan be performed with respect to introducing at least the nucleic acidsencoding the reductive TCA pathway enzymes or proteins absent in thehost organism. Therefore, introduction of one or more encoding nucleicacids into the microbial organisms of the invention such that themodified organism contains a reductive TCA pathway can confer syngasutilization ability.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, fatty alcohol, fattyaldehyde or fatty acid and any of the intermediate metabolites in thefatty alcohol, fatty aldehyde or fatty acid pathway. All that isrequired is to engineer in one or more of the required enzyme or proteinactivities to achieve biosynthesis of the desired compound orintermediate including, for example, inclusion of some or all of thefatty alcohol, fatty aldehyde or fatty acid biosynthetic pathways.Accordingly, the invention provides a non-naturally occurring microbialorganism that produces and/or secretes fatty alcohol, fatty aldehyde orfatty acid when grown on a carbohydrate or other carbon source andproduces and/or secretes any of the intermediate metabolites shown inthe fatty alcohol, fatty aldehyde or fatty acid pathway when grown on acarbohydrate or other carbon source. The fatty alcohol, fatty aldehydeor fatty acid producing microbial organisms of the invention caninitiate synthesis from an intermediate, for example, a 3-ketoacyl-CoA,a 3-hydroxyacyl-CoA, an enoyl-CoA, an acyl-CoA, an acyl-ACP, acetate,acetaldehyde, acetyl-phosphate, oxaloacetate, matate, malonatesemialdehyde, malonate, malonyl-CoA, acetyl-CoA, or citrate.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a fatty alcohol,fatty aldehyde or fatty acid pathway enzyme or protein in sufficientamounts to produce fatty alcohol, fatty aldehyde or fatty acid. It isunderstood that the microbial organisms of the invention are culturedunder conditions sufficient to produce fatty alcohol, fatty aldehyde orfatty acid. Following the teachings and guidance provided herein, thenon-naturally occurring microbial organisms of the invention can achievebiosynthesis of fatty alcohol, fatty aldehyde or fatty acid resulting inintracellular concentrations between about 0.1-200 mM or more.Generally, the intracellular concentration of fatty alcohol, fattyaldehyde or fatty acid is between about 3-150 mM, particularly betweenabout 5-125 mM and more particularly between about 8-100 mM, includingabout 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular concentrationsbetween and above each of these exemplary ranges also can be achievedfrom the non-naturally occurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. publication2009/0047719, filed Aug. 10, 2007. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. Under such anaerobicor substantially anaerobic conditions, the fatty alcohol, fatty aldehydeor fatty acid producers can synthesize fatty alcohol, fatty aldehyde orfatty acid at intracellular concentrations of 5-10 mM or more as well asall other concentrations exemplified herein. It is understood that, eventhough the above description refers to intracellular concentrations,fatty alcohol, fatty aldehyde or fatty acid producing microbialorganisms can produce fatty alcohol, fatty aldehyde or fatty acidintracellularly and/or secrete the product into the culture medium.

Exemplary fermentation processes include, but are not limited to,fed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation; and continuous fermentation and continuousseparation. In an exemplary batch fermentation protocol, the productionorganism is grown in a suitably sized bioreactor sparged with anappropriate gas. Under anaerobic conditions, the culture is sparged withan inert gas or combination of gases, for example, nitrogen, N₂/CO₂mixture, argon, helium, and the like. As the cells grow and utilize thecarbon source, additional carbon source(s) and/or other nutrients arefed into the bioreactor at a rate approximately balancing consumption ofthe carbon source and/or nutrients. The temperature of the bioreactor ismaintained at a desired temperature, generally in the range of 22-37degrees C., but the temperature can be maintained at a higher or lowertemperature depending on the the growth characteristics of theproduction organism and/or desired conditions for the fermentationprocess. Growth continues for a desired period of time to achievedesired characteristics of the culture in the fermenter, for example,cell density, product concentration, and the like. In a batchfermentation process, the time period for the fermentation is generallyin the range of several hours to several days, for example, 8 to 24hours, or 1, 2, 3, 4 or 5 days, or up to a week, depending on thedesired culture conditions. The pH can be controlled or not, as desired,in which case a culture in which pH is not controlled will typicallydecrease to pH 3-6 by the end of the run. Upon completion of thecultivation period, the fermenter contents can be passed through a cellseparation unit, for example, a centrifuge, filtration unit, and thelike, to remove cells and cell debris. In the case where the desiredproduct is expressed intracellularly, the cells can be lysed ordisrupted enzymatically or chemically prior to or after separation ofcells from the fermentation broth, as desired, in order to releaseadditional product. The fermentation broth can be transferred to aproduct separations unit. Isolation of product occurs by standardseparations procedures employed in the art to separate a desired productfrom dilute aqueous solutions. Such methods include, but are not limitedto, liquid-liquid extraction using a water immiscible organic solvent(e.g., toluene or other suitable solvents, including but not limited todiethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride,chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyltertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethylsulfoxide (DMSO), and the like) to provide an organic solution of theproduct, if appropriate, standard distillation methods, and the like,depending on the chemical characteristics of the product of thefermentation process.

In an exemplary fully continuous fermentation protocol, the productionorganism is generally first grown up in batch mode in order to achieve adesired cell density. When the carbon source and/or other nutrients areexhausted, feed medium of the same composition is supplied continuouslyat a desired rate, and fermentation liquid is withdrawn at the samerate. Under such conditions, the product concentration in the bioreactorgenerally remains constant, as well as the cell density. The temperatureof the fermenter is maintained at a desired temperature, as discussedabove. During the continuous fermentation phase, it is generallydesirable to maintain a suitable pH range for optimized production. ThepH can be monitored and maintained using routine methods, including theaddition of suitable acids or bases to maintain a desired pH range. Thebioreactor is operated continuously for extended periods of time,generally at least one week to several weeks and up to one month, orlonger, as appropriate and desired. The fermentation liquid and/orculture is monitored periodically, including sampling up to every day,as desired, to assure consistency of product concentration and/or celldensity. In continuous mode, fermenter contents are constantly removedas new feed medium is supplied. The exit stream, containing cells,medium, and product, are generally subjected to a continuous productseparations procedure, with or without removing cells and cell debris,as desired. Continuous separations methods employed in the art can beused to separate the product from dilute aqueous solutions, includingbut not limited to continuous liquid-liquid extraction using a waterimmiscible organic solvent (e.g., toluene or other suitable solvents,including but not limited to diethyl ether, ethyl acetate,tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane,hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE),dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and thelike), standard continuous distillation methods, and the like, or othermethods well known in the art.

In addition to the culturing and fermentation conditions disclosedherein, growth condition for achieving biosynthesis of fatty alcohol,fatty aldehyde or fatty acid can include the addition of anosmoprotectant to the culturing conditions. In certain embodiments, thenon-naturally occurring microbial organisms of the invention can besustained, cultured or fermented as described herein in the presence ofan osmoprotectant. Briefly, an osmoprotectant refers to a compound thatacts as an osmolyte and helps a microbial organism as described hereinsurvive osmotic stress. Osmoprotectants include, but are not limited to,betaines, amino acids, and the sugar trehalose. Non-limiting examples ofsuch are glycine betaine, praline betaine, dimethylthetin,dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate,pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine andectoine. In one aspect, the osmoprotectant is glycine betaine. It isunderstood to one of ordinary skill in the art that the amount and typeof osmoprotectant suitable for protecting a microbial organism describedherein from osmotic stress will depend on the microbial organism used.The amount of osmoprotectant in the culturing conditions can be, forexample, no more than about 0.1 mM, no more than about 0.5 mM, no morethan about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM,no more than about 2.5 mM, no more than about 3.0 mM, no more than about5.0 mM, no more than about 7.0 mM, no more than about 10 mM, no morethan about 50 mM, no more than about 100 mM or no more than about 500mM.

In some embodiments, the carbon feedstock and other cellular uptakesources such as phosphate, ammonia, sulfate, chloride and other halogenscan be chosen to alter the isotopic distribution of the atoms present infatty alcohol, fatty aldehyde or fatty acid or any fatty alcohol, fattyaldehyde or fatty acid pathway intermediate. The various carbonfeedstock and other uptake sources enumerated above will be referred toherein, collectively, as “uptake sources.” Uptake sources can provideisotopic enrichment for any atom present in the product fatty alcohol,fatty aldehyde or fatty acid or fatty alcohol, fatty aldehyde or fattyacid pathway intermediate, or for side products generated in reactionsdiverging away from a fatty alcohol, fatty aldehyde or fatty acidpathway. Isotopic enrichment can be achieved for any target atomincluding, for example, carbon, hydrogen, oxygen, nitrogen, sulfur,phosphorus, chloride or other halogens.

In some embodiments, the uptake sources can be selected to alter thecarbon-12, carbon-13, and carbon-14 ratios. In some embodiments, theuptake sources can be selected to alter the oxygen-16, oxygen-17, andoxygen-18 ratios. In some embodiments, the uptake sources can beselected to alter the hydrogen, deuterium, and tritium ratios. In someembodiments, the uptake sources can be selected to alter the nitrogen-14and nitrogen-15 ratios. In some embodiments, the uptake sources can beselected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35ratios. In some embodiments, the uptake sources can be selected to alterthe phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In someembodiments, the uptake sources can be selected to alter thechlorine-35, chlorine-36, and chlorine-37 ratios.

In some embodiments, the isotopic ratio of a target atom can be variedto a desired ratio by selecting one or more uptake sources. An uptakesource can be derived from a natural source, as found in nature, or froma man-made source, and one skilled in the art can select a naturalsource, a man-made source, or a combination thereof, to achieve adesired isotopic ratio of a target atom. An example of a man-made uptakesource includes, for example, an uptake source that is at leastpartially derived from a chemical synthetic reaction. Such isotopicallyenriched uptake sources can be purchased commercially or prepared in thelaboratory and/or optionally mixed with a natural source of the uptakesource to achieve a desired isotopic ratio. In some embodiments, atarget atom isotopic ratio of an uptake source can be achieved byselecting a desired origin of the uptake source as found in nature. Forexample, as discussed herein, a natural source can be a biobased derivedfrom or synthesized by a biological organism or a source such aspetroleum-based products or the atmosphere. In some such embodiments, asource of carbon, for example, can be selected from a fossilfuel-derived carbon source, which can be relatively depleted ofcarbon-14, or an environmental or atmospheric carbon source, such asCO₂, which can possess a larger amount of carbon-14 than itspetroleum-derived counterpart.

The unstable carbon isotope carbon-14 or radiocarbon makes up forroughly 1 in 10¹² carbon atoms in the earth's atmosphere and has ahalf-life of about 5700 years. The stock of carbon is replenished in theupper atmosphere by a nuclear reaction involving cosmic rays andordinary nitrogen (¹⁴N). Fossil fuels contain no carbon-14, as itdecayed long ago. Burning of fossil fuels lowers the atmosphericcarbon-14 fraction, the so-called “Suess effect”.

Methods of determining the isotopic ratios of atoms in a compound arewell known to those skilled in the art. Isotopic enrichment is readilyassessed by mass spectrometry using techniques known in the art such asaccelerated mass spectrometry (AMS), Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC), high performance liquid chromatography (HPLC) and/or gaschromatography, and the like.

In the case of carbon, ASTM D6866 was developed in the United States asa standardized analytical method for determining the biobased content ofsolid, liquid, and gaseous samples using radiocarbon dating by theAmerican Society for Testing and Materials (ASTM) International. Thestandard is based on the use of radiocarbon dating for the determinationof a product's biobased content. ASTM D6866 was first published in 2004,and the current active version of the standard is ASTM D6866-11(effective Apr. 1, 2011). Radiocarbon dating techniques are well knownto those skilled in the art, including those described herein.

The biobased content of a compound is estimated by the ratio ofcarbon-14 (¹⁴C) to carbon-12 (¹²C). Specifically, the Fraction Modern(Fm) is computed from the expression: Fm=(S−B)/(M−B), where B, S and Mrepresent the ¹⁴C/¹²C ratios of the blank, the sample and the modernreference, respectively. Fraction Modern is a measurement of thedeviation of the 14C/¹²C ratio of a sample from “Modern.” Modern isdefined as 95% of the radiocarbon concentration (in AD 1950) of NationalBureau of Standards (NBS) Oxalic Acid I (i.e., standard referencematerials (SRM) 4990b) normalized to δ¹³C_(VPDB)=−19 per mil (Olsson,The use of Oxalic acid as a Standard. in, Radiocarbon Variations andAbsolute Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, NewYork (1970)). Mass spectrometry results, for example, measured by ASM,are calculated using the internationally agreed upon definition of 0.95times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalizedto δ¹³C_(VPDB)=−19 per mil. This is equivalent to an absolute (AD 1950)¹⁴C/¹²C ratio of 1.176±0.010×10⁻¹² (Karlen et al., Arkiv Geofysik,4:465-471 (1968)). The standard calculations take into account thedifferential uptake of one isotope with respect to another, for example,the preferential uptake in biological systems of C¹² over C¹³ over C¹⁴,and these corrections are reflected as a Fm corrected for δ¹³.

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of1955 sugar beet. Although there were 1000 lbs made, this oxalic acidstandard is no longer commercially available. The Oxalic Acid IIstandard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of1977 French beet molasses. In the early 1980's, a group of 12laboratories measured the ratios of the two standards. The ratio of theactivity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). Theisotopic ratio of HOx II is −17.8 per mil. ASTM D6866-11 suggests use ofthe available Oxalic Acid II standard SRM 4990 C (Hox2) for the modernstandard (see discussion of original vs. currently available oxalic acidstandards in Mann, Radiocarbon, 25(2):519-527 (1983)). A Fm=0%represents the entire lack of carbon-14 atoms in a material, thusindicating a fossil (for example, petroleum based) carbon source. AFm=100%, after correction for the post-1950 injection of carbon-14 intothe atmosphere from nuclear bomb testing, indicates an entirely moderncarbon source. As described herein, such a “modern” source includesbiobased sources.

As described in ASTM D6866, the percent modern carbon (pMC) can begreater than 100% because of the continuing but diminishing effects ofthe 1950s nuclear testing programs, which resulted in a considerableenrichment of carbon-14 in the atmosphere as described in ASTM D6866-11.Because all sample carbon-14 activities are referenced to a “pre-bomb”standard, and because nearly all new biobased products are produced in apost-bomb environment, all pMC values (after correction for isotopicfraction) must be multiplied by 0.95 (as of 2010) to better reflect thetrue biobased content of the sample. A biobased content that is greaterthan 103% suggests that either an analytical error has occurred, or thatthe source of biobased carbon is more than several years old.

ASTM D6866 quantifies the biobased content relative to the material'stotal organic content and does not consider the inorganic carbon andother non-carbon containing substances present. For example, a productthat is 50% starch-based material and 50% water would be considered tohave a Biobased Content=100% (50% organic content that is 100% biobased)based on ASTM D6866. In another example, a product that is 50%starch-based material, 25% petroleum-based, and 25% water would have aBiobased Content=66.7% (75% organic content but only 50% of the productis biobased). In another example, a product that is 50% organic carbonand is a petroleum-based product would be considered to have a BiobasedContent=0% (50% organic carbon but from fossil sources). Thus, based onthe well known methods and known standards for determining the biobasedcontent of a compound or material, one skilled in the art can readilydetermine the biobased content and/or prepared downstream products thatutilize of the invention having a desired biobased content.

Applications of carbon-14 dating techniques to quantify bio-basedcontent of materials are known in the art (Currie et al., NuclearInstruments and Methods in Physics Research B, 172:281-287 (2000)). Forexample, carbon-14 dating has been used to quantify bio-based content interephthalate-containing materials (Colonna et al., Green Chemistry,13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT)polymers derived from renewable 1,3-propanediol and petroleum-derivedterephthalic acid resulted in Fm values near 30% (i.e., since 3/11 ofthe polymeric carbon derives from renewable 1,3-propanediol and 8/11from the fossil end member terephthalic acid) (Currie et al., supra,2000). In contrast, polybutylene terephthalate polymer derived from bothrenewable 1,4-butanediol and renewable terephthalic acid resulted inbio-based content exceeding 90% (Colonna et al., supra, 2011).

Accordingly, in some embodiments, the present invention provides fattyalcohol, fatty aldehyde or fatty acid or a fatty alcohol, fatty aldehydeor fatty acid pathway intermediate that has a carbon-12, carbon-13, andcarbon-14 ratio that reflects an atmospheric carbon, also referred to asenvironmental carbon, uptake source. For example, in some aspects thefatty alcohol, fatty aldehyde or fatty acid or a fatty alcohol, fattyaldehyde or fatty acid pathway intermediate can have an Fm value of atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98% or as much as 100%.In some such embodiments, the uptake source is CO₂. In some embodiments,the present invention provides fatty alcohol, fatty aldehyde or fattyacid or a fatty alcohol, fatty aldehyde or fatty acid pathwayintermediate that has a carbon-12, carbon-13, and carbon-14 ratio thatreflects petroleum-based carbon uptake source. In this aspect, the fattyalcohol, fatty aldehyde or fatty acid or a fatty alcohol, fatty aldehydeor fatty acid pathway intermediate can have an Fm value of less than95%, less than 90%, less than 85%, less than 80%, less than 75%, lessthan 70%, less than 65%, less than 60%, less than 55%, less than 50%,less than 45%, less than 40%, less than 35%, less than 30%, less than25%, less than 20%, less than 15%, less than 10%, less than 5%, lessthan 2% or less than 1%. In some embodiments, the present inventionprovides fatty alcohol, fatty aldehyde or fatty acid or a fatty alcohol,fatty aldehyde or fatty acid pathway intermediate that has a carbon-12,carbon-13, and carbon-14 ratio that is obtained by a combination of anatmospheric carbon uptake source with a petroleum-based uptake source.Using such a combination of uptake sources is one way by which thecarbon-12, carbon-13, and carbon-14 ratio can be varied, and therespective ratios would reflect the proportions of the uptake sources.

Further, the present invention relates to the biologically producedfatty alcohol, fatty aldehyde or fatty acid or fatty alcohol, fattyaldehyde or fatty acid pathway intermediate as disclosed herein, and tothe products derived therefrom, wherein the fatty alcohol, fattyaldehyde or fatty acid or a fatty alcohol, fatty aldehyde or fatty acidpathway intermediate has a carbon-12, carbon-13, and carbon-14 isotoperatio of about the same value as the CO₂ that occurs in the environment.For example, in some aspects the invention provides bioderived fattyalcohol, fatty aldehyde or fatty acid or a bioderived fatty alcohol,fatty aldehyde or fatty acid intermediate having a carbon-12 versuscarbon-13 versus carbon-14 isotope ratio of about the same value as theCO₂ that occurs in the environment, or any of the other ratios disclosedherein. It is understood, as disclosed herein, that a product can have acarbon-12 versus carbon-13 versus carbon-14 isotope ratio of about thesame value as the CO₂ that occurs in the environment, or any of theratios disclosed herein, wherein the product is generated frombioderived fatty alcohol, fatty aldehyde or fatty acid or a bioderivedfatty alcohol, fatty aldehyde or fatty acid pathway intermediate asdisclosed herein, wherein the bioderived product is chemically modifiedto generate a final product. Methods of chemically modifying abioderived product of fatty alcohol, fatty aldehyde or fatty acid, or anintermediate thereof, to generate a desired product are well known tothose skilled in the art, as described herein. The invention furtherprovides biofuels, chemicals, polymers, surfactants, soaps, detergents,shampoos, lubricating oil additives, fragrances, flavor materials oracrylates having a carbon-12 versus carbon-13 versus carbon-14 isotoperatio of about the same value as the CO₂ that occurs in the environment,wherein the biofuels, chemicals, polymers, surfactants, soaps,detergents, shampoos, lubricating oil additives, fragrances, flavormaterials or acrylates are generated directly from or in combinationwith bioderived fatty alcohol, fatty aldehyde or fatty acid or abioderived fatty alcohol, fatty aldehyde or fatty acid pathwayintermediate as disclosed herein.

Fatty alcohol, fatty aldehyde or fatty acid is a chemical used incommercial and industrial applications. Non-limiting examples of suchapplications include production of biofuels, chemicals, polymers,surfactants, soaps, detergents, shampoos, lubricating oil additives,fragrances, flavor materials and acrylates. Accordingly, in someembodiments, the invention provides biobased biofuels, chemicals,polymers, surfactants, soaps, detergents, shampoos, lubricating oiladditives, fragrances, flavor materials and acrylates comprising one ormore bioderived fatty alcohol, fatty aldehyde or fatty acid orbioderived fatty alcohol, fatty aldehyde or fatty acid pathwayintermediate produced by a non-naturally occurring microorganism of theinvention or produced using a method disclosed herein.

As used herein, the term “bioderived” means derived from or synthesizedby a biological organism and can be considered a renewable resourcesince it can be generated by a biological organism. Such a biologicalorganism, in particular the microbial organisms of the inventiondisclosed herein, can utilize feedstock or biomass, such as, sugars orcarbohydrates obtained from an agricultural, plant, bacterial, or animalsource. Alternatively, the biological organism can utilize atmosphericcarbon. As used herein, the term “biobased” means a product as describedabove that is composed, in whole or in part, of a bioderived compound ofthe invention. A biobased or bioderived product is in contrast to apetroleum derived product, wherein such a product is derived from orsynthesized from petroleum or a petrochemical feedstock.

In some embodiments, the invention provides a biofuel, chemical,polymer, surfactant, soap, detergent, shampoo, lubricating oil additive,fragrance, flavor material or acrylate comprising bioderived fattyalcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol, fattyaldehyde or fatty acid pathway intermediate, wherein the bioderivedfatty alcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol,fatty aldehyde or fatty acid pathway intermediate includes all or partof the fatty alcohol, fatty aldehyde or fatty acid or fatty alcohol,fatty aldehyde or fatty acid pathway intermediate used in the productionof a biofuel, chemical, polymer, surfactant, soap, detergent, shampoo,lubricating oil additive, fragrance, flavor material or acrylate. Forexample, the final biofuel, chemical, polymer, surfactant, soap,detergent, shampoo, lubricating oil additive, fragrance, flavor materialor acrylate can contain the bioderived fatty alcohol, fatty aldehyde orfatty acid, fatty alcohol, fatty aldehyde or fatty acid pathwayintermediate, or a portion thereof that is the result of themanufacturing of the biofuel, chemical, polymer, surfactant, soap,detergent, shampoo, lubricating oil additive, fragrance, flavor materialor acrylate. Such manufacturing can include chemically reacting thebioderived fatty alcohol, fatty aldehyde or fatty acid, or bioderivedfatty alcohol, fatty aldehyde or fatty acid pathway intermediate (e.g.chemical conversion, chemical functionalization, chemical coupling,oxidation, reduction, polymerization, copolymerization and the like)with itself or another compound in a reaction that produces the finalbiofuel, chemical, polymer, surfactant, soap, detergent, shampoo,lubricating oil additive, fragrance, flavor material or acrylate. Thus,in some aspects, the invention provides a biobased biofuel, chemical,polymer, surfactant, soap, detergent, shampoo, lubricating oil additive,fragrance, flavor material or acrylate comprising at least 2%, at least3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98% or100% bioderived fatty alcohol, fatty aldehyde or fatty acid orbioderived fatty alcohol, fatty aldehyde or fatty acid pathwayintermediate as disclosed herein. In some aspects, when the product is abiobased polymer that includes or is obtained from a bioderived fattyalcohol, fatty aldehyde or fatty acid, or or fatty alcohol, fattyaldehyde or fatty acid pathway intermediate described herein, thebiobased polymer can be molded using methods well known in the art.Accordingly, in some embodiments, provided herein is a molded productcomprising the biobased polymer described herein.

Additionally, in some embodiments, the invention provides a compositionhaving a bioderived fatty alcohol, fatty aldehyde or fatty acid, orfatty alcohol, fatty aldehyde or fatty acid pathway intermediatedisclosed herein and a compound other than the bioderived fatty alcohol,fatty aldehyde or fatty acid or fatty alcohol, fatty aldehyde or fattyacid pathway intermediate. For example, in some aspects, the inventionprovides a biobased biofuel, chemical, polymer, surfactant, soap,detergent, shampoo, lubricating oil additive, fragrance, flavor materialor acrylate wherein the fatty alcohol, fatty aldehyde or fatty acid orfatty alcohol, fatty aldehyde or fatty acid pathway intermediate used inits production is a combination of bioderived and petroleum derivedfatty alcohol, fatty aldehyde or fatty acid or fatty alcohol, fattyaldehyde or fatty acid pathway intermediate. For example, a biobased abiofuel, chemical, polymer, surfactant, soap, detergent, shampoo,lubricating oil additive, fragrance, flavor material or acrylate can beproduced using 50% bioderived fatty alcohol, fatty aldehyde or fattyacid and 50% petroleum derived fatty alcohol, fatty aldehyde or fattyacid or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%,95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% ofbioderived/petroleum derived precursors, so long as at least a portionof the product comprises a bioderived product produced by the microbialorganisms disclosed herein. It is understood that methods for producinga biofuel, chemical, polymer, surfactant, soap, detergent, shampoo,lubricating oil additive, fragrance, flavor material or acrylate usingthe bioderived fatty alcohol, fatty aldehyde or fatty acid or bioderivedfatty alcohol, fatty aldehyde or fatty acid pathway intermediate of theinvention are well known in the art.

The invention further provides a composition comprising bioderived fattyalcohol, fatty aldehyde or fatty acid, and a compound other than thebioderived fatty alcohol, fatty aldehyde or fatty acid. The compoundother than the bioderived product can be a cellular portion, forexample, a trace amount of a cellular portion of, or can be fermentationbroth or culture medium, or a purified or partially purified fractionthereof produced in the presence of, a non-naturally occurring microbialorganism of the invention having a fatty alcohol, fatty aldehyde orfatty acid pathway. The composition can comprise, for example, a reducedlevel of a byproduct when produced by an organism having reducedbyproduct formation, as disclosed herein. The composition can comprise,for example, bioderived fatty alcohol, fatty aldehyde or fatty acid, ora cell lysate or culture supernatant of a microbial organism of theinvention.

In certain embodiments, provided herein is a composition comprising abioderived fatty alcohol, fatty aldehyde or fatty acid provided herein,for example, a bioderived fatty alcohol, fatty aldehyde or fatty acidproduced by culturing a non-naturally occurring microbial organismhaving a MI-FAE cycle and/or a MD-FAE cycle in combination with atermination pathway, as provided herein. In some embodiments, thecomposition further comprises a compound other than said bioderivedbioderived fatty alcohol, fatty aldehyde or fatty acid. In certainembodiments, the compound other than said bioderived fatty alcohol,fatty aldehyde or fatty acid is a trace amount of a cellular portion ofa non-naturally occurring microbial organism having a MI-FAE cycleand/or a MD-FAE cycle in combination with a termination pathway.

In some embodiments, provided herein is a biobased product comprising abioderived fatty alcohol, fatty aldehyde or fatty acid provided herein.In certain embodiments, the biobased product is a biofuel, chemical,polymer, surfactant, soap, detergent, shampoo, lubricating oil additive,fragrance, flavor material or acrylate. In certain embodiments, thebiobased product comprises at least 5% bioderived fatty alcohol, fattyaldehyde or fatty acid. In certain embodiments, the biobased productcomprises at least 10% bioderived fatty alcohol, fatty aldehyde or fattyacid. In some embodiments, the biobased product comprises at least 20%bioderived fatty alcohol, fatty aldehyde or fatty acid. In otherembodiments, the biobased product comprises at least 30% bioderivedfatty alcohol, fatty aldehyde or fatty acid. In some embodiments, thebiobased product comprises at least 40% bioderived fatty alcohol, fattyaldehyde or fatty acid. In other embodiments, the biobased productcomprises at least 50% bioderived fatty alcohol, fatty aldehyde or fattyacid. In one embodiment, the biobased product comprises a portion ofsaid bioderived fatty alcohol, fatty aldehyde or fatty acid as arepeating unit. In another embodiment, provided herein is a moldedproduct obtained by molding the biobased product provided herein. Inother embodiments, provided herein is a process for producing a biobasedproduct provided herein, comprising chemically reacting said bioderivedfatty alcohol, fatty aldehyde or fatty acid with itself or anothercompound in a reaction that produces said biobased product. In certainembodiments, provided herein is a polymer comprising or obtained byconverting the bioderived fatty alcohol, fatty aldehyde or fatty acid.In other embodiments, provided herein is a method for producing apolymer, comprising chemically of enzymatically converting thebioderived fatty alcohol, fatty aldehyde or fatty acid to the polymer.In yet other embodiments, provided herein is a composition comprisingthe bioderived fatty alcohol, fatty aldehyde or fatty acid, or a celllysate or culture supernatant thereof.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of fatty alcohol, fatty aldehyde or fatty acid includesanaerobic culture or fermentation conditions. In certain embodiments,the non-naturally occurring microbial organisms of the invention can besustained, cultured or fermented under anaerobic or substantiallyanaerobic conditions. Briefly, an anaerobic condition refers to anenvironment devoid of oxygen. Substantially anaerobic conditionsinclude, for example, a culture, batch fermentation or continuousfermentation such that the dissolved oxygen concentration in the mediumremains between 0 and 10% of saturation. Substantially anaerobicconditions also includes growing or resting cells in liquid medium or onsolid agar inside a sealed chamber maintained with an atmosphere of lessthan 1% oxygen. The percent of oxygen can be maintained by, for example,sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygengas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of fatty alcohol, fatty aldehyde or fattyacid. Exemplary growth procedures include, for example, fed-batchfermentation and batch separation; fed-batch fermentation and continuousseparation, or continuous fermentation and continuous separation. All ofthese processes are well known in the art. Fermentation procedures areparticularly useful for the biosynthetic production of commercialquantities of fatty alcohol, fatty aldehyde or fatty acid. Generally,and as with non-continuous culture procedures, the continuous and/ornear-continuous production of fatty alcohol, fatty aldehyde or fattyacid will include culturing a non-naturally occurring fatty alcohol,fatty aldehyde or fatty acid producing organism of the invention insufficient nutrients and medium to sustain and/or nearly sustain growthin an exponential phase. Continuous culture under such conditions caninclude, for example, growth or culturing for 1 day, 2, 3, 4, 5, 6 or 7days or more. Additionally, continuous culture can include longer timeperiods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms of the invention can be cultured for hours, ifsuitable for a particular application. It is to be understood that thecontinuous and/or near-continuous culture conditions also can includeall time intervals in between these exemplary periods. It is furtherunderstood that the time of culturing the microbial organism of theinvention is for a sufficient period of time to produce a sufficientamount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of fatty alcohol, fatty aldehyde orfatty acid can be utilized in, for example, fed-batch fermentation andbatch separation; fed-batch fermentation and continuous separation, orcontinuous fermentation and continuous separation. Examples of batch andcontinuous fermentation procedures are well known in the art.

In addition to the above fermentation procedures using the fattyalcohol, fatty aldehyde or fatty acid producers of the invention forcontinuous production of substantial quantities of fatty alcohol, fattyaldehyde or fatty acid, the fatty alcohol, fatty aldehyde or fatty acidproducers also can be, for example, simultaneously subjected to chemicalsynthesis and/or enzymatic procedures to convert the product to othercompounds or the product can be separated from the fermentation cultureand sequentially subjected to chemical and/or enzymatic conversion toconvert the product to other compounds, if desired.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of fatty alcohol, fattyaldehyde or fatty acid.

In addition to active and selective enzymes producing fatty alcohols,fatty aldehydes, or fatty acids at high yield, titer and productivity, arobust host organism that can efficiently direct carbon and reducingequivalents to fatty alcohol, fatty aldehyde and fatty acid biosynthesiscan be beneficial. Host modifications described herein are particularlyuseful in combination with selective enzymes described herein that favorformation of the desired fatty alcohol, fatty aldehyde, or fatty acidproduct. Several host modifications described herein entail introducingheterologous enzyme activities into the host organism. Othermodifications involve overexpressing or elevating enzyme activityrelative to wild type levels. Yet other modifications include disruptingendogenous genes or attenuating endogenous enzyme activities.

In one embodiment of the invention, the microbial organisms efficientlydirects carbon and energy sources into production of acetyl-CoA, whichis used as both a primer and extension unit in the MI-FAE cycle. In oneembodiment of the invention, the microbial organisms efficiently directscarbon and energy sources into production of malonyl-CoA, which is usedas both a primer and extension unit in the MD-FAE cycle. In unmodifiedmicrobial organism, fatty alcohol, fatty aldehyde and fatty acidproduction in the cytosol relies on the native cell machinery to providethe necessary precursors. Thus, high concentrations of cytosolicacetyl-CoA and/or malonyl-CoA are desirable for facilitating deploymentof a cytosolic fatty alcohol, fatty aldehyde or fatty acid productionpathway that originates from acetyl-CoA or malonyl-CoA. Metabolicengineering strategies for increasing cytosolic acetyl-CoA andmalonyl-CoA are disclosed herein.

Since many eukaryotic organisms synthesize most of their acetyl-CoA inthe mitochondria during growth on glucose, increasing the availabilityof acetyl-CoA in the cytosol can be obtained by introduction of acytosolic acetyl-CoA biosynthesis pathway. Accordingly, acetyl-CoAbiosynthesis pathways are described herein. In one embodiment, utilizingthe pathways shown in FIG. 2, acetyl-CoA can be synthesized in thecytosol from a pyruvate or threonine precursor. In other embodiment,acetyl-CoA can be synthesized in the cytosol from phosphoenolpyruvate(PEP) or pyruvate (FIG. 3). In yet another embodiment acetyl-CoA can besynthesized in cellular compartments and transported to the cytosol. Forexample, one mechanism involves converting mitochondrial acetyl-CoA to ametabolic intermediate such as citrate or citramalate, transportingthose intermediates to the cytosol, and then regenerating the acetyl-CoA(see FIGS. 4 and 5). Exemplary acetyl-CoA pathways and correspondingenzymes are further described in Examples II-IV.

In another embodiment, increasing cytosolic acetyl-CoA availability forfatty alcohol, fatty aldehyde, or fatty acid biosynthesis is to disruptor attenuate competing enzymes and pathways that utilize acetyl-CoA orits precursors. Exemplary competing enzyme activities include, but arenot limited to, pyruvate decarboxylase, lactate dehydrogenase,short-chain aldehyde and alcohol dehydrogenases, acetate kinase,phosphotransacetylase, glyceraldehyde-3-phosphate dehydrogenases,pyruvate oxidase and acetyl-CoA carboxylase. Exemplary acetyl-CoAconsuming pathways whose disruption or attenuation can improve fattyalcohol, fatty aldehyde, or fatty acid production include themitochondrial TCA cycle, fatty acid biosynthesis, ethanol production andamino acid biosynthesis. These enzymes and pathways are furtherdescribed herein.

Yet another strategy for increasing cytosolic acetyl-CoA production isto increase the pool of CoA available in the cytoplasm. This can beaccomplished by overexpression of CoA biosynthetic enzymes in thecytosol. In particular, expression of pantothenate kinase (EC 2.7.1.33)can be used. This enzyme catalyzes the first step and rate-limitingenzyme of CoA biosynthesis. Exemplary pantothenate kinase variantsresistant to feedback inhibition by CoA are well known in the art (Rocket al, J Bacteriol 185: 3410-5 (2003)) and are described in the belowtable.

Protein Accession # GI number Organism coaA AAC76952 1790409 Escherichiacoli CAB1 NP_010820.3 398366683 Saccharomyces cerevisiae KLLA0C00869gXP_452233.1 50304555 Kluyveromyces lactis YALI0D25476g XP_503275.150551601 Yarrowia lipolytica ANI_1_3272024 XP_001400486.2 317028058Aspergillus niger

Competing enzymes and pathways that divert acyl-CoA substrates fromproduction of fatty alcohols, fatty aldehydes or fatty acids of theinvention can also be attenuated or disrupted. Exemplary enzymes forattenuation include acyltransferases, carnitine shuttle enzymes andnegative regulators of MI-FAE cycle, MD-FAE cycle or termination pathwayenzymes.

Disruption or attenuation of acyltransferases that transfer acylmoieties from CoA to other acceptors such as ACP, glycerol, ethanol andothers, can increase the availability of acyl-CoA for fatty alcohol,fatty aldehyde or fatty acid production. For example, Acyl-CoA:ACPtransacylase (EC 2.3.1.38; 2.3.1.39) enzymes such as fabH (KASIII) of E.coli transfer acyl moieties from CoA to ACP. FabH is active onacetyl-CoA and butyryl-CoA (Prescott et al, Adv. Enzymol. Relat. AreasMol, 36:269-311 (1972)). Acetyl-CoA:ACP transacylase enzymes fromPlasmodium falciparum and Streptomyces avermitillis have beenheterologously expressed in E. coli (Lobo et al, Biochem 40:11955-64(2001)). A synthetic KASIII (FabH) from P. falciparum expressed in afabH-deficient Lactococcus lactis host was able to complement the nativefadH activity (Du et al, AEM 76:3959-66 (2010)). The acetyl-CoA:ACPtransacylase enzyme from Spinacia oleracea accepts other acyl-ACPmolecules as substrates, including butyryl-ACP (Shimakata et al, MethodsEnzym 122:53-9 (1986)). Malonyl-CoA:ACP transacylase enzymes includeFabD of E. coli and Brassica napsus (Verwoert et al, J Bacteriol,174:2851-7 (1992); Simon et al, FEBS Lett 435:204-6 (1998)). FabD of B.napsus was able to complement fabD-deficient E. coli. Themultifunctional eukaryotic fatty acid synthase enzyme complexes(described herein) also catalyze this activity. Other exemplaryacyltransferases include diacylglycerol acyltransferases such as LRO1and DGA1 of S. cerevisiae and DGA1 and DGA2 of Yarrowia lipolytica,glycerolipid acyltransferase enzymes such as plsB of E. coli (GenBank:AAC77011.2, GI:87082362; Heath and Rock, J Bacteriol 180:1425-30(1998)), sterol acyltransferases such as ARE1 and ARE2 of S. cerevisiae,ethanol acyltransferases (EEB1, EHT1), putative acyltransferases(YMR210W) and others.

Protein GenBank ID GI Number Organism fabH AAC74175.1 1787333Escherichia coli fadA NP_824032.1 29829398 Streptomyces avermitillisfabH AAC63960.1 3746429 Plasmodium falciparum Synthetic ACX34097.1260178848 Plasmodium falciparum construct fabH CAL98359.1 124493385Lactococcus lactis fabD AAC74176.1 1787334 Escherichia coli fabDCAB45522.1 5139348 Brassica napsus LRO1 NP_014405.1 6324335Saccharomyces cerevisiae DGA1 NP_014888.1 6324819 Saccharomycescerevisiae DGA1 CAG79269.1 49649549 Yarrowia lipolytica DGA2 XP_504700.150554583 Yarrowia lipolytica ARE1 NP_009978.1 6319896 Saccharomycescerevisiae ARE2 NP_014416.1 6324346 Saccharomyces cerevisiae EEB1NP_015230.1 6325162 Saccharomyces cerevisiae EHT1 NP_009736.3 398365307Saccharomyces cerevisiae YMR210W NP_013937.1 6323866 Saccharomycescerevisiae ALE1 NP_014818.1 6324749 Saccharomyces cerevisiae

Increasing production of fatty alcohols, fatty aldehydes or fatty acidsmay necessitate disruption or attenuation of enzymes involved in thetrafficking of acetyl-CoA and acyl-CoA molecules from the cytosol toother compartments of the organism such as mitochondria, endoplasmicreticulum, proteoliposomes and peroxisomes. In these compartments, theacyl-CoA intermediate can be degraded or used as building blocks tosynthesize fatty acids, cofactors and other byproducts.

Acetyl-CoA and acyl-CoA molecules localized in the cytosol can betransported into other cellular compartments with the aid of the carriermolecule carnitine via carnitine shuttles (van Roermund et al., EMBO J14:3480-86 (1995)). Acyl-carnitine shuttles between cellularcompartments have been characterized in yeasts such as Candida albicans(Strijbis et al, J Biol Chem 285:24335-46 (2010)). In these shuttles,the acyl moiety of acyl-CoA is reversibly transferred to carnitine byacylcarnitine transferase enzymes. Acetylcarnitine can then betransported across the membrane by organelle-specificacylcarnitine/carnitine translocase enzymes. After translocation, theacyl-CoA is regenerated by acetylcarnitine transferase. Enzymes suitablefor disruption or attenuation include carnitine acyltransferase enzymes,acylcarnitine translocases, acylcarnitine carrier proteins and enzymesinvolved in carnitine biosynthesis.

Carnitine acetyltransferase (CAT, EC 2.3.1.7) reversibly links acetylunits from acetyl-CoA to the carrier molecule, carnitine. Candidaalbicans encodes three CAT isozymes: Cat2, Yat1 and Yat2 (Strijbis etal., J Biol Chem 285:24335-46 (2010)). Cat2 is expressed in both themitochondrion and the peroxisomes, whereas Yat1 and Yat2 are cytosolic.The Cat2 transcript contains two start codons that are regulated underdifferent carbon source conditions. The longer transcript contains amitochondrial targeting sequence whereas the shorter transcript istargeted to peroxisomes. Cat2 of Saccharomyces cerevisiae and AcuJ ofAspergillus nidulans employ similar mechanisms of dual localization(Elgersma et al., EMBO J 14:3472-9 (1995); Hynes et al., Euk Cell10:547-55 (2011)). The cytosolic CAT of A. nidulans is encoded by facC.Other exemplary CAT enzymes are found in Rattus norvegicus and Homosapiens (Cordente et al., Biochem 45:6133-41 (2006)). Exemplarycarnitine acyltransferase enzymes (EC 2.3.1.21) are the Cpt1 and Cpt2gene products of Rattus norvegicus (de Vries et al., Biochem 36:5285-92(1997)).

Protein Accession # GI number Organism Cat2 AAN31660.1 23394954 Candidaalbicans Yat1 AAN31659.1 23394952 Candida albicans Yat2 XP_711005.168490355 Candida albicans Cat2 CAA88327.1 683665 Saccharomycescerevisiae Yat1 AAC09495.1 456138 Saccharomyces cerevisiae Yat2NP_010941.1 6320862 Saccharomyces cerevisiae AcuJ CBF69795.1 259479509Aspergillus nidulans FacC AAC82487.1 2511761 Aspergillus nidulans CratAAH83616.1 53733439 Rattus norvegicus Crat P43155.5 215274265 Homosapiens Cpt1 AAB48046.1 1850590 Rattus norvegicus Cpt2 AAB02339.11374784 Rattus norvegicus

Carnitine-acylcarnitine translocases can catalyze the bidirectionaltransport of carnitine and carnitine-fatty acid complexes. The Cact geneproduct provides a mechanism for transporting acyl-carnitine substratesacross the mitochondrial membrane (Ramsay et al Biochim Biophys Acta1546:21-42 (2001)). A similar protein has been studied in humans(Sekoguchi et al., J Biol Chem 278:38796-38802 (2003)). TheSaccharomyces cerevisiae mitochondrial carnitine carrier is Crc1 (vanRoermund et al., supra; Palmieri et al., Biochimica et Biophys Acta1757:1249-62 (2006)). The human carnitine translocase was able tocomplement a Crc1-deficient strain of S. cerevisiae (van Roermund etal., supra). Two additional carnitine translocases found in Drosophilamelanogaster and Caenorhabditis elegans were also able to complementCrc1-deficient yeast (Oey et al., Mol Genet Metab 85:121-24 (2005)).Four mitochondrial carnitine/acetylcarnitine carriers were identified inTrypanosoma brucei based on sequence homology to the yeast and humantransporters (Colasante et al., Mol Biochem Parasit 167:104-117 (2009)).The carnitine transporter of Candida albicans was also identified bysequence homology. An additional mitochondrial carnitine transporter isthe acuH gene product of Aspergillus nidulans, which is exclusivelylocalized to the mitochondrial membrane (Lucas et al., FEMS MicrobiolLett 201:193-8 (2006)).

Protein GenBank ID GI Number Organism Cact P97521.1 2497984 Rattusnorvegicus Cacl NP_001034444.1 86198310 Homo sapiens CaO19.2851XP_715782.1 68480576 Candida albicans Crc1 NP_014743.1 6324674Saccharomyces cerevisiae Dif-1 CAA88283.1 829102 Caenorhabditis eleganscolt CAA73099.1 1944534 Drosophila melanogaster Tb11.02.2960 EAN79492.170833990 Trypanosoma brucei Tb11.03.0870 EAN79007.1 70833505 Trypanosomabrucei Tb11.01.5040 EAN80288.1 70834786 Trypanosoma brucei Tb927.8.5810AAX69329.1 62175181 Trypanosoma brucei acuH CAB44434.1 5019305Aspergillus nidulans

Transport of carnitine and acylcarnitine across the peroxisomal membranehas not been well-characterized. Specific peroxisomal acylcarnitinecarrier proteins in yeasts have not been identified to date. However,mitochonidrial carnitine translocases can also function in theperoxisomal transport of carnitine and acetylcarnitine. Experimentalevidence suggests that the OCTN3 protein of Mus musculus is aperoxisomal carnitine/acylcarnitine translocase.

Yet another possibility is that acyl-CoA or acyl-carnitine aretransported across the peroxisomal or mitochondrial membranes by anacyl-CoA transporter such as the Pxa1 and Pxa2 ABC transporter ofSaccharomyces cerevisiae or the ALDP ABC transporter of Homo sapiens(van Roermund et al., FASEB J22:4201-8 (2008)). Pxa1 and Pxa2 (Pat1 andPat2) form a heterodimeric complex in the peroxisomal membrane andcatalyze the ATP-dependent transport of fatty acyl-CoA esters into theperoxisome (Verleur et al., Eur J Biochem 249: 657-61 (1997)). Themutant phenotype of a pxa1/pxa2 deficient yeast can be rescued byheterologous expression of ALDP, which was shown to transport a range ofacyl-CoA substrates (van Roermund et al., FASEB J22:4201-8 (2008)).Deletion of the Pxa12 transport system, in tandem with deletion of theperoxisomal fatty acyl-CoA synthetase (Faa2) abolished peroxisomalbeta-oxidation in S. cerevisiae. Yet another strategy for reducingtransport of pathway intermediates or products into the peroxisome is toattenuate or eliminate peroxisomal function, by interfering with systemsinvolved in peroxisomal biogenesis. An exemplary target is Pex10 ofYarrowia lipolytica and homologs.

Protein Accession # GI number Organism OCTN3 BAA78343.1 4996131 Musmusculus Pxa1 AAC49009.1 619668 Saccharomyces cerevisiae Pxa2 AAB51597.11931633 Saccharomyces cerevisiae Faa2 NP_010931.3 398364331Saccharomyces cerevisiae ALDP NP_000024.2 7262393 Homo sapiens Pex10BAA99413.1 9049374 Yarrowia lipolytica

Carnitine biosynthetic pathway enzymes are also suitable candidates fordisruption or attenuation. In Candida albicans, for example, carnitineis synthesized from trimethyl-L-lysine in four enzymatic steps (Strijbiset al., FASEB J 23:2349-59 (2009)). The carnitine pathway precursor,trimethyllysine (TML), is produced during protein degradation. TMLdioxygenase (CaO13.4316) hydroxylates TML to form3-hydroxy-6-N-trimethyllysine. A pyridoxal-5′-phosphate dependentaldolase (CaO19.6305) then cleaves HTML into4-trimethylaminobutyraldehyde. The 4-trimethylaminobutyraldehyde issubsequently oxidized to 4-trimethylaminobutyrate by a dehydrogenase(CaO19.6306). In the final step, 4-trimethylaminobutyrate ishydroxylated to form carnitine by the gene product of CaO19.7131. Fluxthrough the carnitine biosynthesis pathway is limited by theavailability of the pathway substrate and very low levels of carnitineseem to be sufficient for normal carnitine shuttle activity (Strejbis etal., IUBMB Life 62:357-62 (2010)).

Protein Accession # GI number Organism CaO19.4316 XP_720623.1 68470755Candida albicans CaO19.6305 XP_711090.1 68490151 Candida albicansCaO19.6306 XP_711091.1 68490153 Candida albicans CaO19.7131 XP_715182.168481628 Candida albicans

Carbon flux towards production of fatty alcohols, fatty aldehydes orfatty acids can be improved by deleting or attenuating competingpathways. Typical fermentation products of yeast include ethanol,glycerol and CO₂. The elimination or reduction of these byproducts canbe accomplished by approaches described herein. For example, carbon lossdue to respiration can be reduced. Other potential byproducts includelactate, acetate, formate, fatty acids and amino acids.

The conversion of acetyl-CoA into ethanol can be detrimental to theproduction of fatty alcohols, fatty aldehyes or fatty acids because theconversion process can draw away both carbon and reducing equivalentsfrom the MI-FAE cycle, MD-FAE cycle and termination pathway. Ethanol canbe formed from pyruvate in two enzymatic steps catalyzed by pyruvatedecarboxylase and ethanol dehydrogenase. Saccharomyces cerevisiae hasthree pyruvate decarboxylases (PDC1, PDC5 and PDC6). PDC1 is the majorisozyme and is strongly expressed in actively fermenting cells. PDC5also functions during glycolytic fermentation, but is expressed only inthe absence of PDC1 or under thiamine limitating conditions. PDC6functions during growth on nonfermentable carbon sources. Deleting PDC1and PDC5 can reduce ethanol production significantly; however thesedeletions can lead to mutants with increased PDC6 expression. Deletionof all three eliminates ethanol formation completely but also can causea growth defect because of inability of the cells to form sufficientacetyl-CoA for biomass formation. This, however, can be overcome byevolving cells in the presence of reducing amounts of C2 carbon source(ethanol or acetate) (van Maris et al, AEM 69:2094-9 (2003)). It hasalso been reported that deletion of the positive regulator PDC2 ofpyruvate decarboxylases PDC1 and PDC5, reduced ethanol formation to ˜10%of that made by wild-type (Hohmann et al, Mol Gen Genet 241:657-66(1993)). Protein sequences and identifiers of PDC enzymes are listed inExample II.

Alternatively, alcohol dehydrogenases that convert acetaldehyde intoethanol and/or other short chain alcohol dehydrogenases can be disruptedor attenuated to provide carbon and reducing equivalents for the MI-FAEcycle, MD-FAE or termination pathway. To date, seven alcoholdehydrogenases, ADHI-ADHVII, have been reported in S. cerevisiae (deSmidt et al, FEMS Yeast Res 8:967-78 (2008)). ADH1 (GI:1419926) is thekey enzyme responsible for reducing acetaldehyde to ethanol in thecytosol under anaerobic conditions. It has been reported that a yeaststrain deficient in ADH1 cannot grow anaerobically because an activerespiratory chain is the only alternative path to regenerate NADH andlead to a net gain of ATP (Drewke et al, J Bacteriol 172:3909-17(1990)). This enzyme is an ideal candidate for downregulation to limitethanol production. ADH2 is severely repressed in the presence ofglucose. In K. lactis, two NAD-dependent cytosolic alcoholdehydrogenases have been identified and characterized. These genes alsoshow activity for other aliphatic alcohols. The genes ADH1 (GI:113358)and ADHII (GI:51704293) are preferentially expressed in glucose-growncells (Bozzi et al, Biochim Biophys Acta 1339:133-142 (1997)). Cytosolicalcohol dehydrogenases are encoded by ADH1 (GI:608690) in C. albicans,ADH1 (GI:3810864) in S. pombe, ADH1 (GI:5802617) in Y. lipolytica, ADH1(GI:2114038) and ADHII (GI:2143328) in Pichia stipitis orScheffersomyces stipitis (Passoth et al, Yeast 14:1311-23 (1998)).Candidate alcohol dehydrogenases are shown the table below.

Protein GenBank ID GI number Organism SADH BAA24528.1 2815409 Candidaparapsilosis ADH1 NP_014555.1 6324486 Saccharomyces cerevisiae s288cADH2 NP_014032.1 6323961 Saccharomyces cerevisiae s288c ADH3 NP_013800.16323729 Saccharomyces cerevisiae s288c ADH4 NP_011258.2 269970305Saccharomyces cerevisiae s288c ADH5 (SFA1) NP_010113.1 6320033Saccharomyces cerevisiae s288c ADH6 NP_014051.1 6323980 Saccharomycescerevisiae s288c ADH7 NP_010030.1 6319949 Saccharomyces cerevisiae s288cadhP CAA44614.1 2810 Kluyveromyces lactis ADH1 P20369.1 113358Kluyveromyces lactis ADH2 CAA45739.1 2833 Kluyveromyces lactis ADH3P49384.2 51704294 Kluyveromyces lactis ADH1 CAA57342.1 608690 Candidaalbicans ADH2 CAA21988.1 3859714 Candida albicans SAD XP_712899.168486457 Candida albicans ADH1 CAA21782.1 3810864 Schizosaccharomycespombe ADH1 AAD51737.1 5802617 Yarrowia lipolytica ADH2 AAD51738.15802619 Yarrowia lipolytica ADH3 AAD51739.1 5802621 Yarrowia lipolyticaAlcB AAX53105.1 61696864 Aspergillus niger ANI_1_282024 XP_001399347.1145231748 Aspergillus niger ANI_1_126164 XP_001398574.2 317037131Aspergillus niger ANI_1_1756104 XP_001395505.2 317033815 Aspergillusniger ADH2 CAA73827.1 2143328 Scheffersomyces stipitis

Attenuation or disruption of one or more glycerol-3-phosphatase orglycerol-3-phosphate (G3P) dehydrogenase enzymes can eliminate or reducethe formation of glycerol, and thereby conserving carbon and reducingequivalents for production of fatty alcohols, fatty aldehydes or fattyacids.

G3P phosphatase catalyzes the hydrolysis of G3P to glycerol. Enzymeswith this activity include the glycerol-1-phosphatase (EC 3.1.3.21)enzymes of Saccharomyces cerevisiae (GPP1 and GPP2), Candida albicansand Dunaleilla parva (Popp et al, Biotechnol Bioeng 100:497-505 (2008);Fan et al, FEMS Microbiol Lett 245:107-16 (2005)). The D. parva gene hasnot been identified to date. These and additional G3P phosphataseenzymes are shown in the table below.

Protein GenBank ID GI Number Organism GPP1 DAA08494.1 285812595Saccharomyces cerevisiae GPP2 NP_010984.1 6320905 Saccharomycescerevisiae GPP1 XP_717809.1 68476319 Candida albicans KLLA0C08217gXP_452565.1 50305213 Kluyveromyces lactis KLLA0C11143g XP_452697.150305475 Kluyveromyces lactis ANI_1_380074 XP_001392369.1 145239445Aspergillus niger ANI_1_444054 XP_001390913.2 317029125 Aspergillusniger

S. cerevisiae has three G3P dehydrogenase enzymes encoded by GPD1 andGDP2 in the cytosol and GUT2 in the mitochondrion. GPD2 is known toencode the enzyme responsible for the majority of the glycerol formationand is responsible for maintaining the redox balance under anaerobicconditions. GPD1 is primarily responsible for adaptation of S.cerevisiae to osmotic stress (Bakker et al., FEMS Microbiol Rev 24:15-37(2001)). Attenuation of GPD1, GPD2 and/or GUT2 will reduce glycerolformation. GPD1 and GUT2 encode G3P dehydrogenases in Yarrowialipolytica (Beopoulos et al, AEM 74:7779-89 (2008)). GPD1 and GPD2encode for G3P dehydrogenases in S. pombe. Similarly, G3P dehydrogenaseis encoded by CTRG_(—)02011 in Candida tropicalis and a gene representedby GI:20522022 in Candida albicans.

Protein GenBank ID GI number Organism GPD1 CAA98582.1 1430995Saccharomyces cerevisiae GPD2 NP_014582.1 6324513 Saccharomycescerevisiae GUT2 NP_012111.1 6322036 Saccharomyces cerevisiae GPD1CAA22119.1 6066826 Yarrowia lipolytica GUT2 CAG83113.1 49646728 Yarrowialipolytica GPD1 CAA22119.1 3873542 Schizosaccharomyces pombe GPD2CAA91239.1 1039342 Schizosaccharomyces pombe ANI_1_786014 XP_001389035.2317025419 Aspergillus niger ANI_1_1768134 XP_001397265.1 145251503Aspergillus niger KLLA0C04004g XP_452375.1 50304839 Kluyveromyces lactisCTRG_02011 XP_002547704.1 255725550 Candida tropicalis GPD1 XP_714362.168483412 Candida albicans GPD2 XP_713824.1 68484586 Candida albicans

Enzymes that form acid byproducts such as acetate, formate and lactatecan also be attenuated or disrupted. Such enzymes include acetatekinase, phosphotransacetylase and pyruvate oxidase. Disruption orattenuation of pyruvate formate lyase and formate dehydrogenase couldlimit formation of formate and carbon dioxide. These enzymes aredescribed in further detail in Example II.

Alcohol dehydrogenases that convert pyruvate to lactate are alsocandidates for disruption or attenuation. Lactate dehydrogenase enzymesinclude ldhA of E. coli and ldh from Ralstonia eutropha (Steinbuchel andSchlegel, Eur. J. Biochem. 130:329-334 (1983)). Other alcoholdehydrogenases listed above may also exhibit LDH activity.

Protein GenBank ID GI number Organism ldhA NP_415898.1 16129341Escherichia coli Ldh YP_725182.1 113866693 Ralstonia eutropha

Tuning down activity of the mitochondrial pyruvate dehydrogenase complexwill limit flux into the mitochondrial TCA cycle. Under anaerobicconditions and in conditions where glucose concentrations are high inthe medium, the capacity of this mitochondrial enzyme is very limitedand there is no significant flux through it. However, in someembodiments, this enzyme can be disrupted or attenuated to increasefatty alcohol, fatty aldehyde or fatty acid production. Exemplarypyruvate dehydrogenase genes include PDB1, PDA1, LAT1 and LPD1.Accession numbers and homologs are listed in Example II.

Another strategy for reducing flux into the TCA cycle is to limittransport of pyruvate into the mitochondria by tuning down or deletingthe mitochondrial pyruvate carrier. Transport of pyruvate into themitochondria in S. cerevisiae is catalyzed by a heterocomplex encoded byMPC1 and MPC2 (Herzig et al, Science 337:93-6 (2012); Bricker et al,Science 337:96-100 (2012)). S. cerevisiae encodes five other putativemonocarboxylate transporters (MCH1-5), several of which may be localizedto the mitochondrial membrane (Makuc et al, Yeast 18:1131-43 (2001)).NDT1 is another putative pyruvate transporter, although the role of thisprotein is disputed in the literature (Todisco et al, J Biol Chem20:1524-31 (2006)). Exemplary pyruvate and monocarboxylate transportersare shown in the table below:

Protein GenBank ID GI number Organism MPC1 NP_011435.1 6321358Saccharomyces cerevisiae MPC2 NP_012032.1 6321956 Saccharomycescerevisiae MPC1 XP_504811.1 50554805 Yarrowia lipolytica MPC2XP_501390.1 50547841 Yarrowia lipolytica MPC1 XP_719951.1 68471816Candida albicans MPC2 XP_716190.1 68479656 Candida albicans MCH1NP_010229.1 6320149 Saccharomyces cerevisiae MCH2 NP_012701.2 330443640Saccharomyces cerevisiae MCH3 NP_014274.1 6324204 Saccharomycescerevisiae MCH5 NP_014951.2 330443742 Saccharomyces cerevisiae NDT1NP_012260.1 6322185 Saccharomyces cerevisiae ANI_1_1592184XP_001401484.2 317038471 Aspergillus niger CaJ7_0216 XP_888808.177022728 Candida albicans YALI0E16478g XP_504023.1 50553226 Yarrowialipolytica KLLA0D14036g XP_453688.1 50307419 Kluyveromyces lactis

Disruption or attenuation of enzymes that synthesize malonyl-CoA andfatty acids can increase the supply of carbon available for fattyalcohol, fatty aldehyde or fatty acid biosynthesis from acetyl-CoA.Exemplary enzymes for disruption or attenuation include fatty acidsynthase, acetyl-CoA carboxylase, biotin:apoenzyme ligase, acyl carrierprotein, thioesterase, acyltransferases, ACP malonyltransferase, fattyacid elongase, acyl-CoA synthetase, acyl-CoA transferase and acyl-CoAhydrolase.

Another strategy to reduce fatty acid biosynthesis is expression oroverexpression of regulatory proteins which repress fatty acid forminggenes. Acetyl-CoA carboxylase (EC 6.4.1.2) catalyzes the first step offatty acid biosynthesis in many organisms: the ATP-dependentcarboxylation of acetyl-CoA to malonyl-CoA. This enzyme utilizes biotinas a cofactor. Exemplary ACC enzymes are encoded by accABCD of E. coli(Davis et al, J Biol Chem 275:28593-8 (2000)), ACCT of Saccharomycescerevisiae and homologs (Sumper et al, Methods Enzym 71:34-7 (1981)).The mitochondrial acetyl-CoA carboxylase of S. cerevisiae is encoded byHFA1. Acetyl-CoA carboxylase holoenzyme formation requires attachment ofbiotin by a biotin:apoprotein ligase such as BPL1 of S. cerevisiae.

Protein GenBank ID GI Number Organism ACC1 CAA96294.1 1302498Saccharomyces cerevisiae KLLA0F06072g XP_455355.1 50310667 Kluyveromyceslactis ACC1 XP_718624.1 68474502 Candida albicans YALI0C11407pXP_501721.1 50548503 Yarrowia lipolytica ANI_1_1724104 XP_001395476.1145246454 Aspergillus niger accA AAC73296.1 1786382 Escherichia coliaccB AAC76287.1 1789653 Escherichia coli accC AAC76288.1 1789654Escherichia coli accD AAC75376.1 1788655 Escherichia coli HFA1NP_013934.1 6323863 Saccharomyces cerevisiae BPL1 NP_010140.1 6320060Saccharomyces cerevisiae

Proteins participating in the synthesis of fatty acids are shown below.The fatty acid synthase enzyme complex of yeast is composed of twomultifunctional subunits, FAS1 and FAS2, which together catalyze the netconversion of acetyl-CoA and malonyl-CoA to fatty acids (Lomakin et al,Cell 129: 319-32 (2007)). Additional proteins associated withmitochondrial fatty acid synthesis include OAR1, Mctl, ETR1, ACP1 andPPT2. ACP1 is the mitochondrial acyl carrier protein and PPT2 encodes aphosphopantetheine transferase, which pantetheinylates mitochondrial ACPand is required for fatty acid biosynthesis in the mitochondria (Stuibleet al, J Biol Chem: 273: 22334-9 (1998)). A non-genetic strategy forreducing activity of fatty acid synthases is to add an inhibitor such ascerulenin. Global regulators of lipid biosynthesis can also be alteredto tune down endogenous fatty acid biosynthesis pathways duringproduction of long chain alcohols or related products. An exemplaryglobal regulator is SNF1 of Yarrowia lipolytica and Saccharomycescerevisiae.

Protein GenBank ID GI Number Organism FAS1 NP_012739.1 6322666Saccharomyces cerevisiae FAS2 NP_015093.1 6325025 Saccharomycescerevisiae FAS1 XP_451653.1 50303423 Kluyveromyces lactis FAS2XP_452914.1 50305907 Kluyveromyces lactis FAS1 XP_716817.1 68478392Candida albicans FAS2 XP_723014.1 68465892 Candida albicans FAS1XP_500912.1 50546885 Yarrowia lipolytica FAS2 XP_501096.1 50547253Yarrowia lipolytica FAS1 XP_001393490.2 317031809 Aspergillus niger FAS2XP_001388458.1 145228299 Aspergillus niger OAR1 NP_012868.1 6322795Saccharomyces cerevisiae MCT1 NP_014864.4 398365823 Saccharomycescerevisiae ETR1 NP_009582.1 6319500 Saccharomyces cerevisiae ACP1NP_012729.1 6322656 Saccharomyces cerevisiae PPT2 NP_015177.2 37362701Saccharomyces cerevisiae SNF1 CAG80498.1 49648180 Yarrowia lipolyticaSNF1 P06782.1 134588 Saccharomyces cerevisiae

Disruption or attenuation of elongase enzymes which convert acyl-CoAsubstrates to longer-chain length fatty acids can also be used toincrease fatty alcohol, fatty aldehyde or fatty acid production.Elongase enzymes are found in compartments such as the mitochondria,endoplasmic reticulum, proteoliposomes and peroxisomes. For example,some yeast such as S. cerevisiae are able to synthesize long-chain fattyacids of chain length C16 and higher via a mitochondrial elongase whichaccepts exogenous or endogenous acyl-CoA substrates (Bessoule et al,FEBS Lett 214: 158-162 (1987)). This system requires ATP for activity.The endoplasmic reticulum also has an elongase system for synthesizingvery long chain fatty acids (C18+) from acyl-CoA substrates of varyinglengths (Kohlwein et al, Mol Cell Biol 21:109-25 (2001)). Genes involvedin this system include TSC13, ELO2 and ELO3. ELO1 catalyzes theelongation of C12 acyl-CoAs to C16-C18 fatty acids.

Protein Accession # GI number Organism ELO2 NP_009963.1 6319882Saccharomyces cerevisiae ELO3 NP_013476.3 398366027 Saccharomycescerevisiae TSC13 NP_010269.1 6320189 Saccharomyces cerevisiae ELO1NP_012339.1 6322265 Saccharomyces cerevisiae

Native enzymes converting acyl-CoA pathway intermediates to acidbyproducts can also reduce fatty alcohol, fatty aldehyde or fatty acidyield. For example, CoA hydrolases, transferases and synthetases can acton acyl-CoA intermediates to form short-, medium- or long chain acids.Disruption or attenuation of endogenous CoA hydrolases, CoA transferasesand/or reversible CoA synthetases can be used to increase fatty alcohol,fatty aldehyde or fatty acid yield. Exemplary enzymes are shown in thetable below.

Protein GenBank ID GI number Organism Tes1 NP_012553.1 6322480Saccharomyces cerevisiae s288c ACH1 NP_009538.1 6319456 Saccharomycescerevisiae s288c EHD3 NP_010321.1 6320241 Saccharomyces cerevisiae s288cYALI0F14729p XP_505426.1 50556036 Yarrowia lipolytica YALI0E30965pXP_504613.1 50554409 Yarrowia lipolytica KLLA0E16523g XP_454694.150309373 Kluyveromyces lactis KLLA0E10561g XP_454427.1 50308845Kluyveromyces lactis ACH1 P83773.2 229462795 Candida albicansCaO19.10681 XP_714720.1 68482646 Candida albicans ANI_1_318184XP_001401512.1 145256774 Aspergillus niger ANI_1_1594124 XP_001401252.2317035188 Aspergillus niger tesB NP_414986.1 16128437 Escherichia colitesB NP_355686.2 159185364 Agrobacterium tumefaciens atoA 2492994P76459.1 Escherichia coli atoD 2492990 P76458.1 Escherichia coli

Enzymes that favor the degradation of products, MI-FAE cycleintermediates, MD-FAE cycle intermediates or termination pathwayintermediates can also be disrupted or attenuated. Examples includealdehyde dehydrogenases, aldehyde decarbonylases, oxidative alcoholdehydrogenases, and irreversible fatty acyl-CoA degrading enzymes.

For production of fatty alcohols, fatty aldehydes or fatty acids of theinvention, deletion or attenuation of non-specific aldehydedehydrogenases can improve yield. For production of fatty acids,expression of such an enzyme may improve product formation. Such enzymescan, for example, convert acetyl-CoA into acetaldehyde, fatty aldehydesto fatty acids, or fatty alcohols to fatty acids. Acylating aldehydedehydrogenase enzymes are described in Example I. Acid-forming aldehydedehydrogenase are described in Examples III and IX.

The pathway enzymes that favor the reverse direction can also bedisrupted or attenuated, if they are detrimental to fatty alcohol, fattyaldehyde or fatty acid production. An example is long chain alcoholdehydrogenases (EC 1.1.1.192) that favor the oxidative direction.Exemplary long chain alcohol dehydrogenases are ADH1 and ADH2 ofGeobacillus thermodenitrificans, which oxidize alcohols up to a chainlength of C30 (Liu et al, Physiol Biochem 155:2078-85 (2009)). These andother exemplary fatty alcohol dehydrogenase enzymes are listed inExamples I and II. If an alcohol-forming acyl-CoA reductase is utilizedfor fatty alcohol, fatty aldehyde or fatty acid biosynthesis, deletionof endogenous fatty alcohol dehydrogenases will substantially reducebackflux.

Beta-oxidation enzymes may be reversible and operate in the direction ofacyl-CoA synthesis. However, if they are irreversible or stronglyfavored in the degradation direction they are candidates for disruptionor attenuation. An enzyme that fall into this category includes FOX2 ofS. cerevisiae, a multifunctional enzyme with 3-hydroxyacyl-CoAdehydrogenase and enoyl-CoA hydratase activity (Hiltunen et al, J BiolChem 267: 6646-6653 (1992)). Additional genes include degradativethiolases such as POT1 and acyl-CoA dehydrogenases that utilizecofactors other than NAD(P)H (EG. EC 1.3.8.-) such as fadE of E. coli.

Protein GenBank ID GI Number Organism POT1 NP_012106.1 6322031Saccharomyces cerevisiae FOX2 NP_012934.1 6322861 Saccharomycescerevisiae fadE AAC73325.2 87081702 Escherichia coli

Fatty acyl-CoA oxidase enzymes such as POX1 of S. cerevisiae catalyzethe oxygen-dependent oxidation of fatty acyl-CoA substrates. Enzymeswith this activity can be disrupted or attenuated, if they are expressedunder fatty alcohol, fatty aldehyde or fatty acid producing conditions.POX1 (EC 1.3.3.6) genes and homologs are shown in the table below. POX1is subject to regulation by OAF1, which also activates genes involved inperoxisomal beta-oxidation, organization and biogenesis (Luo et al, JBiol Chem 271:12068-75 (1996)). Regulators with functions similar toOAF1, and peroxisomal fatty acid transporters PXA1 and PXA2 are alsocandidates for deletion.

Protein GenBank ID GI Number Organism POX1 NP_011310.1 6321233Saccharomyces cerevisiae OAF1 NP_009349.3 330443370 Saccharomycescerevisiae PXA1 NP_015178.1 6325110 Saccharomyces cerevisiae PXA2NP_012733.1 6322660 Saccharomyces cerevisiae YALI0F10857g XP_505264.150555712 Yarrowia lipolytica YALI0D24750p XP_503244.1 50551539 Yarrowialipolytica YALI0E32835p XP_504703.1 50554589 Yarrowia lipolyticaYALI0E06567p XP_503632.1 50552444 Yarrowia lipolytica YALI0E27654pXP_504475.1 50554133 Yarrowia lipolytica YALI0C23859p XP_502199.150549457 Yarrowia lipolytica POX XP_455532.1 50311017 Kluyveromyceslactis POX104 XP_721610.1 68468582 Candida albicans POX105 XP_717995.168475844 Candida albicans POX102 XP_721613.1 68468588 Candida albicans

Another candidate for disruption or attenuation is an acyl-CoA bindingprotein. The acyl binding protein ACB1 of S. cerevisiae, for example,binds acyl-CoA esters and shuttles them to acyl-CoA utilizing processes(Schjerling et al, J Biol Chem 271: 22514-21 (1996)). Deletion of thisprotein did not impact growth rate and lead to increased accumulation oflonger-chain acyl-CoA molecules. Acyl-CoA esters are involved in diversecellular processes including lipid biosynthesis and homeostatis, signaltransduction, growth regulation and cell differentiation (Rose et al,PNAS USA 89: 11287-11291 (1992)).

Protein GenBank ID GI Number Organism ACB1 P31787.3 398991 Saccharomycescerevisiae KLLA0B05643g XP_451787.2 302309983 Kluyveromyces lactisYALI0E23185g XP_002143080.1 210076210 Yarrowia lipolytica ANI_1_1084034XP_001390082.1 145234867 Aspergillus niger

To achieve high yields of fatty alcohols, fatty aldehydes or fattyacids, it is desirable that the host organism can supply the cofactorsrequired by the MI-FAE cycle, MD-FAE and/or the termination pathway insufficient quantities. In several organisms, in particular eukaryoticorganisms, such as several Saccharomyces, Kluyveromyces, Candida,Aspergillus, and Yarrowia species, NADH is more abundant than NADPH inthe cytosol as it is produced in large quantities by glycolysis. NADHcan be made even more abundant by converting pyruvate to acetyl-CoA bymeans of heterologous or native NAD-dependant enzymes such asNAD-dependant pyruvate dehydrogenase, NAD-dependant formatedehydrogenase, NADH:ferredoxin oxidoreductase, or NAD-dependantacylating acetylaldehyde dehydrogenase in the cytosol. Given theabundance of NADH in the cytosol of most organisms, it can be beneficialfor all reduction steps of the MI-FAE cycle, MD-FAE cycle and/orterminatio pathway to accept NADH as the reducing agent preferentiallyover other reducing agents such as NADPH. High yields of fatty alcohols,fatty aldehydes or fatty acids can thus be accomplished by, forexample: 1) identifying and implementing endogenous or exogenous MI-FAEcycle, MD-FAE cycle and/or termination pathway enzymes with a strongerpreference for NADH than other reducing equivalents such as NADPH; 2)attenuating one or more endogenous MI-FAE cycle, MD-FAE cycle ortermination pathway enzymes that contribute NADPH-dependant reductionactivity; 3) altering the cofactor specificity of endogenous orexogenous MI-FAE cycle, MD-FAE cycle or termination pathway enzymes sothat they have a stronger preference for NADH than their naturalversions; or 4) altering the cofactor specificity of endogenous orexogenous MI-FAE cycle, MD-FAE cycle or termination pathway enzymes sothat they have a weaker preference for NADPH than their naturalversions.

Strategies for engineering NADH-favoring MI-FAE cycle, MD-FAE cycleand/or termination pathways are described in further detail in ExampleV. Methods for changing the cofactor specificity of an enzyme are wellknown in the art, and an example is described in Example VI.

If one or more of the MI-FAE cycle, MD-FAE cycle and/or terminationpathway enzymes utilizes NADPH as the cofactor, it can be beneficial toincrease the production of NADPH in the host organism. In particular, ifthe MI-FAE cycle, MD-FAE cycle and/or termination pathway is present inthe cytosol of the host organism, methods for increasing NADPHproduction in the cytosol can be beneficial. Several approaches forincreasing cytosolic production of NADPH can be implemented includingchanneling an increased amount of flux through the oxidative branch ofthe pentose phosphate pathway relative to wild-type, channeling anincreased amount of flux through the Entner Doudoroff pathway relativeto wild-type, introducing a soluble or membrane-bound transhydrogenaseto convert NADH to NADPH, or employing NADP-dependant versions of thefollowing enzymes: phosphorylating or non-phosphorylatingglyceraldehyde-3-phosphate dehydrogenase, pyruvate dehydrogenase,formate dehydrogenase, or acylating acetylaldehyde dehydrogenase. Theseactivities can be augmented by disrupting or attenuating nativeNAD-dependant enzymes including glyceraldehyde-3-phosphatedehydrogenase, pyruvate dehydrogenase, formate dehydrogenase, oracylating acetylaldehyde dehydrogenase. Strategies for engineeringincreased NADPH availability are described in Example VII.

Synthesis of fatty alcohols, fatty aldehyes or fatty acids in thecytosol can be dependent upon the availability of sufficient carbon andreducing equivalents. Therefore, without being bound to any particulartheory of operation, increasing the redox ratio of NAD(P)H to NAD(P) canhelp drive the MI-FAE cycle, MD-FAE cycle and/or termination pathway inthe forward direction. Methods for increasing the redox ratio of NAD(P)Hto NAD(P) include limiting respiration, attenuating or disruptingcompeting pathways that produce reduced byproducts such as ethanol andglycerol, attenuating or eliminating the use of NADH by NADHdehydrogenases, and attenuating or eliminating redox shuttles betweencompartments.

One exemplary method to provide an increased number of reducingequivalents, such as NAD(P)H, for enabling the formation of fattyalcohols, fatty aldehydes or fatty acids is to constrain the use of suchreducing equivalents during respiration. Respiration can be limited by:reducing the availability of oxygen, attenuating NADH dehydrogenasesand/or cytochrome oxidase activity, attenuating G3P dehydrogenase,and/or providing excess glucose to Crabtree positive organisms.

Restricting oxygen availability by culturing the non-naturally occurringeukaryotic organisms in a fermenter is one example for limitingrespiration and thereby increasing the ratio of NAD(P)H to NAD(P). Theratio of NAD(P)H/NAD(P) increases as culture conditions become moreanaerobic, with completely anaerobic conditions providing the highestratios of the reduced cofactors to the oxidized ones. For example, ithas been reported that the ratio of NADH/NAD=0.02 in aerobic conditionsand 0.75 in anaerobic conditions in E. coli (de Graes et al, J Bacteriol181:2351-57 (1999)).

Respiration can also be limited by reducing expression or activity ofNADH dehydrogenases and/or cytochrome oxidases in the cell under aerobicconditions. In this case, respiration can be limited by the capacity ofthe electron transport chain. Such an approach has been used to enableanaerobic metabolism of E. coli under completely aerobic conditions(Portnoy et al, AEM 74:7561-9 (2008)). S. cerevisiae can oxidizecytosolic NADH directly using external NADH dehydrogenases, encoded byNDE1 and NDE2. One such NADH dehydrogenase in Yarrowia lipolytica isencoded by NDH2 (Kerscher et al, J Cell Sci 112:2347-54 (1999)). Theseand other NADH dehydrogenase enzymes are listed in the table below.

Protein GenBank ID GI number Organism NDE1 NP_013865.1 6323794Saccharomyces cerevisiae s288c NDE2 NP_010198.1 6320118 Saccharomycescerevisiae s288c NDH2 AJ006852.1 3718004 Yarrowia lipolyticaANI_1_610074 XP_001392541.2 317030427 Aspergillus niger ANI_1_2462094XP_001394893.2 317033119 Aspergillus niger KLLA0E21891g XP_454942.150309857 Kluyveromyces lactis KLLA0C06336g XP_452480.1 50305045Kluyveromyces lactis NDE1 XP_720034.1 68471982 Candida albicans NDE2XP_717986.1 68475826 Candida albicans

Cytochrome oxidases of Saccharomyces cerevisiae include the COX geneproducts. COX1-3 are the three core subunits encoded by themitochondrial genome, whereas COX4-13 are encoded by nuclear genes.Attenuation or disruption of any of the cytochrome genes results in adecrease or block in respiratory growth (Hermann and Funes, Gene354:43-52 (2005)). Cytochrome oxidase genes in other organisms can beinferred by sequence homology.

Protein GenBank ID GI number Organism COX1 CAA09824.1 4160366Saccharomyces cerevisiae s288c COX2 CAA09845.1 4160387 Saccharomycescerevisiae s288c COX3 CAA09846.1 4160389 Saccharomyces cerevisiae s288cCOX4 NP_011328.1 6321251 Saccharomyces cerevisiae s288c COX5ANP_014346.1 6324276 Saccharomyces cerevisiae s288c COX5B NP_012155.16322080 Saccharomyces cerevisiae s288c COX6 NP_011918.1 6321842Saccharomyces cerevisiae s288c COX7 NP_013983.1 6323912 Saccharomycescerevisiae s288c COX8 NP_013499.1 6323427 Saccharomyces cerevisiae s288cCOX9 NP_010216.1 6320136 Saccharomyces cerevisiae s288c COX12NP_013139.1 6323067 Saccharomyces cerevisiae s288c COX13 NP_011324.16321247 Saccharomyces cerevisiae s288c

Cytosolic NADH can also be oxidized by the respiratory chain via the G3Pdehydrogenase shuttle, consisting of cytosolic NADH-linked G3Pdehydrogenase and a membrane-bound G3P:ubiquinone oxidoreductase. Thedeletion or attenuation of G3P dehydrogenase enzymes will also preventthe oxidation of NADH for respiration. Enzyme candidates encoding theseenzymes are described herein.

Additionally, in Crabtree positive organisms, fermentative metabolismcan be achieved in the presence of excess of glucose. For example, S.cerevisiae makes ethanol even under aerobic conditions. The formation ofethanol and glycerol can be reduced/eliminated and replaced by theproduction of fatty alcohol, fatty aldehyde or fatty acid in a Crabtreepositive organism by feeding excess glucose to the Crabtree positiveorganism. In another aspect, provided herein is a method for producingfatty alcohols, fatty aldehydes or fatty acids, comprising culturing anon-naturally occurring eukaryotic organism under conditions and for asufficient period of time to produce fatty alcohol, fatty aldehyde orfatty acid, wherein the eukaryotic organism is a Crabtree positiveorganism that comprises at least one exogenous nucleic acid encoding aMI-FAE cycle, MD-FAE cycle and/or termination pathway enzyme and whereineukaryotic organism is in a culture medium comprising excess glucose.

Preventing formation of reduced fermentation byproducts will increasethe availability of both carbon and reducing equivalents for fattyalcohol, fatty aldehyde or fatty acid production. The two key reducedbyproducts under anaerobic and microaerobic conditions are ethanol andglycerol. Ethanol is typically formed from pyruvate in two enzymaticsteps catalyzed by pyruvate decarboxylase and ethanol dehydrogenase.Glycerol is formed from the glycolytic intermediate dihydroxyacetonephosphate by the enzymes glycerol-3-phosphate dehydrogenase andglycerol-3-phosphate phosphatase. Attenuation of one or more of theseenzyme activities will increase the yield of fatty alcohols, fattyaldehydes or fatty acids. Strain engineering strategies for reducing oreliminating ethanol and glycerol formation are described herein.

Yeast such as S. cerevisiae can produce glycerol to allow forregeneration of NAD(P) under anaerobic conditions. Another way to reduceor eliminate glycerol production is by oxygen-limited cultivation(Bakker et al, supra). Glycerol formation only sets in when the specificoxygen uptake rates of the cells decrease below the rate that isrequired to reoxidize the NADH formed in biosynthesis.

In addition to the redox sinks listed above, malate dehydrogenase canpotentially draw away reducing equivalents when it functions in thereductive direction. Several redox shuttles believed to be functional inS. cerevisiae utilize this enzyme to transfer reducing equivalentsbetween the cytosol and the mitochondria. This transfer of redox can beprevented by attenuating malate dehydrogenase and/or malic enzymeactivity. The redox shuttles that can be blocked by the attenuation ofmdh include (i) malate-asparate shuttle, (ii) malate-oxaloacetateshuttle, and (iii) malate-pyruvate shuttle. Genes encoding malatedehydrogenase and malic enzymes are listed in the table below.

Protein GenBank ID GI Number Organism MDH1 NP_012838.1 6322765Saccharomyces cerevisiae MDH2 NP_014515.2 116006499 Saccharomycescerevisiae MDH3 NP_010205.1 6320125 Saccharomyces cerevisiae MAE1NP_012896.1 6322823 Saccharomyces cerevisiae MDH1 XP_722674.1 68466384Candida albicans MDH2 XP_718638.1 68474530 Candida albicans MAE1XP_716669.1 68478574 Candida albicans KLLA0F25960g XP_456236.1 50312405Kluyveromyces lactis KLLA0E18635g XP_454793.1 50309563 Kluyveromyceslactis KLLA0E07525g XP_454288.1 50308571 Kluyveromyces lactisYALI0D16753p XP_502909.1 50550873 Yarrowia lipolytica YALI0E18634pXP_504112.1 50553402 Yarrowia lipolytica ANI_1_268064 XP_001391302.1145237310 Aspergillus niger ANI_1_12134 XP_001396546.1 145250065Aspergillus niger ANI_1_22104 XP_001395105.2 317033225 Aspergillus niger

Overall, disruption or attenuation of the aforementioned sinks for redoxeither individually or in combination with the other redox sinks caneliminate or lower the use of reducing power for respiration orbyproduct formation. It has been reported that the deletion of theexternal NADH dehydrogenases (NDE1 and NDE2) and the mitochondrial G3Pdehydrogenase (GUT2) almost completely eliminates cytosolic NAD+regeneration in S. cerevisiae (Overkamp et al, J Bacteriol 182:2823-30(2000)).

Microorganisms of the invention produce fatty alcohols, fatty aldehydesor fatty acids and optionally secrete the fatty alcohols, fattyaldehydes or fatty acis into the culture medium. S. cerevisiae, Yarrowialipolytica and E. coli harboring heterologous fatty alcohol formingactivities accululated fatty alcohols intracellularly; however fattyalcohols were not detected in the culture medium (Behrouzian et al,United States Patent Application 20100298612). The introduction of fattyacyl-CoA reductase enzymes with improved activity resulted in higherlevels of fatty alcohol secreted into the culture media. Alternately,introduction of a fatty alcohol, fatty aldehyde or fatty acidtransporter or transport system can improve extracellular accumulationof fatty alcohols, fatty aldehydes or fatty acids. Exemplarytransporters are listed in the table below.

Protein GenBank ID GI Number Organism Fatp NP_524723.2 24583463Drosophila melanogaster AY161280.1: AAN73268.1 34776949 Rhodococcus 45 .. . 1757 erythropolis acrA CAF23274.1 46399825 Candidatus Protochlamydiaamoebophila acrB CAF23275.1 46399826 Candidatus Protochlamydiaamoebophila CER5 AY734542.1 52354013 Arabidopsis thaliana AmiS2 JC54917449112 Rhodococcus sp. ANI_1_1160064 XP_001391993.1 145238692Aspergillus niger YALI0E16016g XP_504004.1 50553188 Yarrowia lipolytica

Thus, in some embodiments, the invention provides a non-naturallyoccurring microbial organism as disclosed herein having one or more genedisruptions, wherein the one or more gene disruptions occur inendogenous genes encoding proteins or enzymes involved in: nativeproduction of ethanol, glycerol, acetate, formate, lactate, CO₂, fattyacids, or malonyl-CoA by said microbial organism; transfer of pathwayintermediates to cellular compartments other than the cytosol; or nativedegradation of a MI-FAE cycle intermediate, a MD-FAE cycle intermediateor a termination pathway intermediate by the microbial organism, the oneor more gene disruptions confer increased production of a fatty alcohol,fatty aldehyde or fatty acid in the microbial organism. Accordingly, theprotein or enzyme can be a fatty acid synthase, an acetyl-CoAcarboxylase, a biotin:apoenzyme ligase, an acyl carrier protein, athioesterase, an acyltransferase, an ACP malonyltransferase, a fattyacid elongase, an acyl-CoA synthetase, an acyl-CoA transferase, anacyl-CoA hydrolase, a pyruvate decarboxylase, a lactate dehydrogenase,an alcohol dehydrogenase, an acid-forming aldehyde dehydrogenases, anacetate kinase, a phosphotransacetylase, a pyruvate oxidase, aglycerol-3-phosphate dehydrogenase, a glycerol-3-phosphate phosphatase,a mitochondrial pyruvate carrier, a peroxisomal fatty acid transporter,a peroxisomal acyl-CoA transporter, a peroxisomalcarnitine/acylcarnitine transferase, an acyl-CoA oxidase, or an acyl-CoAbinding protein. In some aspects, the one or more gene disruptionsinclude a deletion of the one or more genes.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein, wherein one or more enzymes ofthe MI-FAE cycle, the MD-FAE cycle or the termination pathwaypreferentially react with an NADH cofactor or have reduced preferencefor reacting with an NAD(P)H cofactor. For example, the one or moreenzymes of the MI-FAE cycle can be a 3-ketoacyl-CoA reductase or anenoyl-CoA reductase. For the termination pathway, the one or moreenzymes can be an acyl-CoA reductase (aldehyde forming), an alcoholdehydrogenase, an acyl-CoA reductase (alcohol forming), an aldehydedecarbonylase, an acyl-ACP reductase, an aldehyde dehydrogenase (acidforming) or a carboxylic acid reductase.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein having one or more genedisruptions in genes encoding proteins or enzymes that result in anincreased ratio of NAD(P)H to NAD(P) present in the cytosol of themicrobial organism following the disruptions. Accordingly, the geneencoding a protein or enzyme that results in an increased ratio ofNAD(P)H to NAD(P) present in the cytosol of the microbial organismfollowing the disruptions can be an NADH dehydrogenase, a cytochromeoxidase, a G3P dehydrogenase, G3P phosphatase, an alcohol dehydrogenase,a pyruvate decarboxylase, an aldehyde dehydrogenase (acid forming), alactate dehydrogenase, a glycerol-3-phosphate dehydrogenase, aglycerol-3-phosphate:quinone oxidoreductase, a malic enzyme and a malatedehydrogenase. In some aspects, the one or more gene disruptions includea deletion of the one or more genes.

In some embodiments, the non-naturally occurring microbial organism ofthe invention is Crabtree positive and is in culture medium comprisingexcess glucose. In such conditions, as described herein, the microbialorganism can result in increasing the ratio of NAD(P)H to NAD(P) presentin the cytosol of the microbial organism.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein having at least one exogenousnucleic acid encoding an extracellular transporter or an extracellulartransport system for a fatty alcohol, fatty aldehyde or fatty acid ofthe invention.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein, wherein one or more endogenousenzymes involved in: native production of ethanol, glycerol, acetate,formate, lactate, CO₂, fatty acids, or malonyl-CoA by said microbialorganism; transfer of pathway intermediates to cellular compartmentsother than the cytosol; or native degradation of a MI-FAE cycleintermediate, a MD-FAE cycle intermediate or a termination pathwayintermediate by said microbial organism, has attenuated enzyme activityor expression levels. Accordingly, the endogenous enzyme can be a fattyacid synthase, an acetyl-CoA carboxylase, a biotin:apoenzyme ligase, anacyl carrier protein, a thioesterase, an acyltransferase, an ACPmalonyltransferase, a fatty acid elongase, an acyl-CoA synthetase, anacyl-CoA transferase, an acyl-CoA hydrolase, a pyruvate decarboxylase, alactate dehydrogenase, an alcohol dehydrogenase, an acid-formingaldehyde dehydrogenases, an acetate kinase, a phosphotransacetylase, apyruvate oxidase, a glycerol-3-phosphate dehydrogenase, aglycerol-3-phosphate phosphatase, a mitochondrial pyruvate carrier, aperoxisomal fatty acid transporter, a peroxisomal acyl-CoA transporter,a peroxisomal carnitine/acylcarnitine transferase, an acyl-CoA oxidase,or an acyl-CoA binding protein.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein, wherein one or more endogenousenzymes involved in the oxidation of NAD(P)H or NADH, has attenuatedenzyme activity or expression levels. Accordingly, the one or moreendogenous enzymes can be a NADH dehydrogenase, a cytochrome oxidase, aG3P dehydrogenase, G3P phosphatase, an alcohol dehydrogenase, a pyruvatedecarboxylase, an aldehyde dehydrogenase (acid forming), a lactatedehydrogenase, a glycerol-3-phosphate dehydrogenase, aglycerol-3-phosphate:quinone oxidoreductase, a malic enzyme and a malatedehydrogenase.

The non-naturally occurring microbal organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

In the case of gene disruptions, a particularly useful stable geneticalteration is a gene deletion. The use of a gene deletion to introduce astable genetic alteration is particularly useful to reduce thelikelihood of a reversion to a phenotype prior to the geneticalteration. For example, stable growth-coupled production of abiochemical can be achieved, for example, by deletion of a gene encodingan enzyme catalyzing one or more reactions within a set of metabolicmodifications. The stability of growth-coupled production of abiochemical can be further enhanced through multiple deletions,significantly reducing the likelihood of multiple compensatoryreversions occurring for each disrupted activity.

Also provided is a method of producing a non-naturally occurringmicrobial organisms having stable growth-coupled production of fattyalcohol, fatty aldehyde or fatty acid. The method can includeidentifying in silico a set of metabolic modifications that increaseproduction of fatty alcohol, fatty aldehyde or fatty acid, for example,increase production during exponential growth; genetically modifying anorganism to contain the set of metabolic modifications that increaseproduction of fatty alcohol, fatty aldehyde or fatty acid, and culturingthe genetically modified organism. If desired, culturing can includeadaptively evolving the genetically modified organism under conditionsrequiring production of fatty alcohol, fatty aldehyde or fatty acid. Themethods of the invention are applicable to bacterium, yeast and fungusas well as a variety of other cells and microorganism, as disclosedherein.

Thus, the invention provides a non-naturally occurring microbialorganism comprising one or more gene disruptions that confer increasedproduction of fatty alcohol, fatty aldehyde or fatty acid. In oneembodiment, the one or more gene disruptions confer growth-coupledproduction of fatty alcohol, fatty aldehyde or fatty acid, and can, forexample, confer stable growth-coupled production of fatty alcohol, fattyaldehyde or fatty acid. In another embodiment, the one or more genedisruptions can confer obligatory coupling of fatty alcohol, fattyaldehyde or fatty acid production to growth of the microbial organism.Such one or more gene disruptions reduce the activity of the respectiveone or more encoded enzymes.

The non-naturally occurring microbial organism can have one or more genedisruptions included in a gene encoding a enzyme or protein disclosedherein. As disclosed herein, the one or more gene disruptions can be adeletion. Such non-naturally occurring microbial organisms of theinvention include bacteria, yeast, fungus, or any of a variety of othermicroorganisms applicable to fermentation processes, as disclosedherein.

Thus, the invention provides a non-naturally occurring microbialorganism, comprising one or more gene disruptions, where the one or moregene disruptions occur in genes encoding proteins or enzymes where theone or more gene disruptions confer increased production of fattyalcohol, fatty aldehyde or fatty acid in the organism. The production offatty alcohol, fatty aldehyde or fatty acid can be growth-coupled or notgrowth-coupled. In a particular embodiment, the production of fattyalcohol, fatty aldehyde or fatty acid can be obligatorily coupled togrowth of the organism, as disclosed herein.

The invention provides non naturally occurring microbial organismshaving genetic alterations such as gene disruptions that increaseproduction of fatty alcohol, fatty aldehyde or fatty acid, for example,growth-coupled production of fatty alcohol, fatty aldehyde or fattyacid. Product production can be, for example, obligatorily linked to theexponential growth phase of the microorganism by genetically alteringthe metabolic pathways of the cell, as disclosed herein. The geneticalterations can increase the production of the desired product or evenmake the desired product an obligatory product during the growth phase.Metabolic alterations or transformations that result in increasedproduction and elevated levels of fatty alcohol, fatty aldehyde or fattyacid biosynthesis are exemplified herein. Each alteration corresponds tothe requisite metabolic reaction that should be functionally disrupted.Functional disruption of all reactions within one or more of thepathwyas can result in the increased production of fatty alcohol, fattyaldehyde or fatty acid by the engineered strain during the growth phase.

Each of these non-naturally occurring alterations result in increasedproduction and an enhanced level of fatty alcohol, fatty aldehyde orfatty acid production, for example, during the exponential growth phaseof the microbial organism, compared to a strain that does not containsuch metabolic alterations, under appropriate culture conditions.Appropriate conditions include, for example, those disclosed herein,including conditions such as particular carbon sources or reactantavailabilities and/or adaptive evolution.

Given the teachings and guidance provided herein, those skilled in theart will understand that to introduce a metabolic alteration such asattenuation of an enzyme, it can be necessary to disrupt the catalyticactivity of the one or more enzymes involved in the reaction.Alternatively, a metabolic alteration can include disrupting expressionof a regulatory protein or cofactor necessary for enzyme activity ormaximal activity. Furthermore, genetic loss of a cofactor necessary foran enzymatic reaction can also have the same effect as a disruption ofthe gene encoding the enzyme. Disruption can occur by a variety ofmethods including, for example, deletion of an encoding gene orincorporation of a genetic alteration in one or more of the encodinggene sequences. The encoding genes targeted for disruption can be one,some, or all of the genes encoding enzymes involved in the catalyticactivity. For example, where a single enzyme is involved in a targetedcatalytic activity, disruption can occur by a genetic alteration thatreduces or eliminates the catalytic activity of the encoded geneproduct. Similarly, where the single enzyme is multimeric, includingheteromeric, disruption can occur by a genetic alteration that reducesor destroys the function of one or all subunits of the encoded geneproducts. Destruction of activity can be accomplished by loss of thebinding activity of one or more subunits required to form an activecomplex, by destruction of the catalytic subunit of the multimericcomplex or by both. Other functions of multimeric protein associationand activity also can be targeted in order to disrupt a metabolicreaction of the invention. Such other functions are well known to thoseskilled in the art. Similarly, a target enzyme activity can be reducedor eliminated by disrupting expression of a protein or enzyme thatmodifies and/or activates the target enzyme, for example, a moleculerequired to convert an apoenzyme to a holoenzyme. Further, some or allof the functions of a single polypeptide or multimeric complex can bedisrupted according to the invention in order to reduce or abolish thecatalytic activity of one or more enzymes involved in a reaction ormetabolic modification of the invention. Similarly, some or all ofenzymes involved in a reaction or metabolic modification of theinvention can be disrupted so long as the targeted reaction is reducedor eliminated.

Given the teachings and guidance provided herein, those skilled in theart also will understand that an enzymatic reaction can be disrupted byreducing or eliminating reactions encoded by a common gene and/or by oneor more orthologs of that gene exhibiting similar or substantially thesame activity. Reduction of both the common gene and all orthologs canlead to complete abolishment of any catalytic activity of a targetedreaction. However, disruption of either the common gene or one or moreorthologs can lead to a reduction in the catalytic activity of thetargeted reaction sufficient to promote coupling of growth to productbiosynthesis. Exemplified herein are both the common genes encodingcatalytic activities for a variety of metabolic modifications as well astheir orthologs. Those skilled in the art will understand thatdisruption of some or all of the genes encoding a enzyme of a targetedmetabolic reaction can be practiced in the methods of the invention andincorporated into the non-naturally occurring microbial organisms of theinvention in order to achieve the increased production of fatty alcohol,fatty aldehyde or fatty acid or growth-coupled product production.

Given the teachings and guidance provided herein, those skilled in theart also will understand that enzymatic activity or expression can beattenuated using well known methods. Reduction of the activity or amountof an enzyme can mimic complete disruption of a gene if the reductioncauses activity of the enzyme to fall below a critical level that isnormally required for a pathway to function. Reduction of enzymaticactivity by various techniques rather than use of a gene disruption canbe important for an organism's viability. Methods of reducing enzymaticactivity that result in similar or identical effects of a genedisruption include, but are not limited to: reducing gene transcriptionor translation; destabilizing mRNA, protein or catalytic RNA; andmutating a gene that affects enzyme activity or kinetics (See, Sambrooket al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold SpringHarbor Laboratory, New York (2001); and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1999). Natural or imposed regulatory controls can also accomplishenzyme attenuation including: promoter replacement (See, Wang et al.,Mol. Biotechnol. 52(2):300-308 (2012)); loss or alteration oftranscription factors (Dietrick et al., Annu. Rev. Biochem. 79:563-590(2010); and Simicevic et al., Mol. Biosyst. 6(3):462-468 (2010));introduction of inhibitory RNAs or peptides such as siRNA, antisenseRNA, RNA or peptide/small-molecule binding aptamers, ribozymes,aptazymes and riboswitches (Wieland et al., Methods 56(3):351-357(2012); O'Sullivan, Anal. Bioanal. Chem. 372(1):44-48 (2002); and Lee etal., Curr. Opin. Biotechnol. 14(5):505-511 (2003)); and addition ofdrugs or other chemicals that reduce or disrupt enzymatic activity suchas an enzyme inhibitor, an antibiotic or a target-specific drug.

One skilled in the art will also understand and recognize thatattenuation of an enzyme can be done at various levels. For example, atthe gene level, a mutation causing a partial or complete null phenotype,such as a gene disruption, or a mutation causing epistatic geneticeffects that mask the activity of a gene product (Miko, Nature Education1(1) (2008)), can be used to attenuate an enzyme. At the gene expressionlevel, methods for attenuation include: coupling transcription to anendogenous or exogenous inducer, such as isopropylthio-β-galactoside(IPTG), then adding low amounts of inducer or no inducer during theproduction phase (Donovan et al., J. Ind. Microbiol. 16(3):145-154(1996); and Hansen et al., Curr. Microbiol. 36(6):341-347 (1998));introducing or modifying a positive or a negative regulator of a gene;modify histone acetylation/deacetylation in a eukaryotic chromosomalregion where a gene is integrated (Yang et al., Curr. Opin. Genet. Dev.13(2):143-153 (2003) and Kurdistani et al., Nat. Rev. Mol. Cell Biol.4(4):276-284 (2003)); introducing a transposition to disrupt a promoteror a regulatory gene (Bleykasten-Brosshans et al., C. R. Biol.33(8-9):679-686 (2011); and McCue et al., PLoS Genet. 8(2):e1002474(2012)); flipping the orientation of a transposable element or promoterregion so as to modulate gene expression of an adjacent gene (Wang etal., Genetics 120(4):875-885 (1988); Hayes, Annu Rev. Genet. 37:3-29(2003); in a diploid organism, deleting one allele resulting in loss ofheterozygosity (Daigaku et al., Mutation Research/Fundamental andMolecular Mechanisms of Mutagenesis 600(1-2)177-183 (2006)); introducingnucleic acids that increase RNA degradation (Houseley et al., Cell,136(4):763-776 (2009); or in bacteria, for example, introduction of atransfer-messenger RNA (tmRNA) tag, which can lead to RNA degradationand ribosomal stalling (Sunohara et al., RNA 10(3):378-386 (2004); andSunohara et al., J. Biol. Chem. 279:15368-15375 (2004)). At thetranslational level, attenuation can include: introducing rare codons tolimit translation (Angov, Biotechnol. J. 6(6):650-659 (2011));introducing RNA interference molecules that block translation (Castel etal., Nat. Rev. Genet. 14(2):100-112 (2013); and Kawasaki et al., Curr.Opin. Mol. Ther. 7(2):125-131 (2005); modifying regions outside thecoding sequence, such as introducing secondary structure into anuntranslated region (UTR) to block translation or reduce efficiency oftranslation (Ringnér et al., PLoS Comput. Biol. 1(7):e72 (2005)); addingRNAase sites for rapid transcript degradation (Pasquinelli, Nat. Rev.Genet. 13(4):271-282 (2012); and Arraiano et al., FEMS Microbiol. Rev.34(5):883-932 (2010); introducing antisense RNA oligomers or antisensetranscripts (Nashizawa et al., Front. Biosci. 17:938-958 (2012));introducing RNA or peptide aptamers, ribozymes, aptazymes, riboswitches(Wieland et al., Methods 56(3):351-357 (2012); O'Sullivan, Anal.Bioanal. Chem. 372(1):44-48 (2002); and Lee et al., Curr. Opin.Biotechnol. 14(5):505-511 (2003)); or introducing translationalregulatory elements involving RNA structure that can prevent or reducetranslation that can be controlled by the presence or absence of smallmolecules (Araujo et al., Comparative and Functional Genomics, ArticleID 475731, 8 pages (2012)). At the level of enzyme localization and/orlongevity, enzyme attenuation can include: adding a degradation tag forfaster protein turnover (Hochstrasser, Annual Rev. Genet. 30:405-439(1996); and Yuan et al., PLoS One 8(4):e62529 (2013)); or adding alocalization tag that results in the enzyme being secreted or localizedto a subcellular compartment in a eukaryotic cell, where the enzymewould not be able to react with its normal substrate (Nakai et al.Genomics 14(4):897-911 (1992); and Russell et al., J. Bact.189(21)7581-7585 (2007)). At the level of post-translational regulation,enzyme attenuation can include: increasing intracellular concentrationof known inhibitors; or modifying post-translational modified sites(Mann et al., Nature Biotech. 21:255-261 (2003)). At the level of enzymeactivity, enzyme attenuation can include: adding an endogenous or anexogenous inhibitor, such as an enzyme inhibitor, an antibiotic or atarget-specific drug, to reduce enzyme activity; limiting availabilityof essential cofactors, such as vitamin B12, for an enzyme that requiresthe cofactor; chelating a metal ion that is required for enzymeactivity; or introducing a dominant negative mutation. The applicabilityof a technique for attenuation described above can depend upon whether agiven host microbial organism is prokaryotic or eukaryotic, and it isunderstand that a determination of what is the appropriate technique fora given host can be readily made by one skilled in the art.

In some embodiments, microaerobic designs can be used based on thegrowth-coupled formation of the desired product. To examine this,production cones can be constructed for each strategy by firstmaximizing and, subsequently minimizing the product yields at differentrates of biomass formation feasible in the network. If the rightmostboundary of all possible phenotypes of the mutant network is a singlepoint, it implies that there is a unique optimum yield of the product atthe maximum biomass formation rate possible in the network. In othercases, the rightmost boundary of the feasible phenotypes is a verticalline, indicating that at the point of maximum biomass the network canmake any amount of the product in the calculated range, including thelowest amount at the bottommost point of the vertical line. Such designsare given a low priority.

The fatty alcohol, fatty aldehyde or fatty acid-production strategiesidentified in the various tables disclosed herein can be disrupted toincrease production of fatty alcohol, fatty aldehyde or fatty acid.Accordingly, the invention also provides a non-naturally occurringmicrobial organism having metabolic modifications coupling fattyalcohol, fatty aldehyde or fatty acid production to growth of theorganism, where the metabolic modifications includes disruption of oneor more genes selected from the genes encoding proteins and/or enzymesshown in the various tables disclosed herein.

Each of the strains can be supplemented with additional deletions if itis determined that the strain designs do not sufficiently increase theproduction of fatty alcohol, fatty aldehyde or fatty acid and/or couplethe formation of the product with biomass formation. Alternatively, someother enzymes not known to possess significant activity under the growthconditions can become active due to adaptive evolution or randommutagenesis. Such activities can also be knocked out. However, the listof gene deletion disclosed herein allows the construction of strainsexhibiting high-yield production of fatty alcohol, fatty aldehyde orfatty acid, including growth-coupled production of fatty alcohol, fattyaldehyde or fatty acid.

In some embodiments, the invention provides a method for producing acompound of Formula (I):

wherein R₁ is C₁₋₂₄ linear alkyl; R₂ is CH₂OH, CHO, or COOH; R₃ is H,OH, or oxo (═O); and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four, comprising culturing anon-naturally occurring microbial organism described herein underconditions and for a sufficient period of time to produce the compoundof Formula (I), wherein the non-naturally occurring microbial organismhas one or more gene disruptions, wherein the one or more genedisruptions occur in endogenous genes encoding proteins or enzymesinvolved in: native production of ethanol, glycerol, acetate, formate,lactate, CO₂, fatty acids, or malonyl-CoA by said microbial organism;transfer of pathway intermediates to cellular compartments other thanthe cytosol; or native degradation of a MI-FAE cycle intermediate, aMD-FAE cycle intermediate or a termination pathway intermediate by themicrobial organism, the one or more gene disruptions confer increasedproduction of a fatty alcohol, fatty aldehyde or fatty acid in themicrobial organism. Accordingly, the protein or enzyme can be a fattyacid synthase, an acetyl-CoA carboxylase, a biotin:apoenzyme ligase, anacyl carrier protein, a thioesterase, an acyltransferases, an ACPmalonyltransferase, a fatty acid elongase, an acyl-CoA synthetase, anacyl-CoA transferase, an acyl-CoA hydrolase, a pyruvate decarboxylase, alactate dehydrogenase, an alcohol dehydrogenase, an acid-formingaldehyde dehydrogenases, an acetate kinase, a phosphotransacetylase, apyruvate oxidase, a glycerol-3-phosphate dehydrogenase, aglycerol-3-phosphate phosphatase, a mitochondrial pyruvate carrier, aperoxisomal fatty acid transporters, a peroxisomal acyl-CoAtransporters, a peroxisomal carnitine/acylcarnitine transferases, anacyl-CoA oxidase, or an acyl-CoA binding protein. In some aspects, theone or more gene disruptions include a deletion of the one or moregenes.

In some embodiments, the invention provides a method for producing afatty alcohol, fatty aldehyde or fatty acid using a non-naturallyoccurring microbial organism as described herein, wherein one or moreenzymes of the MI-FAE cycle, MD-FAE cycle or the termination pathwaypreferentially react with an NADH cofactor or have reduced preferencefor reacting with an NAD(P)H cofactor. For example, the one or moreenzymes of the MI-FAE cycle or MD-FAE cycle can be a 3-ketoacyl-CoAreductase or an enoyl-CoA reductase. For the termination pathway, theone or more enzymes can be an acyl-CoA reductase (aldehyde forming), analcohol dehydrogenase, an acyl-CoA reductase (alcohol forming), analdehyde decarbonylase, an acyl-ACP reductase, an aldehyde dehydrogenase(acid forming) or a carboxylic acid reductase.

In some embodiments, the invention provides a method for producing afatty alcohol, fatty aldehyde or fatty acid using a non-naturallyoccurring microbial organism as described herein having one or more genedisruptions in genes encoding proteins or enzymes that result in anincreased ratio of NAD(P)H to NAD(P) present in the cytosol of themicrobial organism following the disruptions. Accordingly, the geneencoding a protein or enzyme that results in an increased ratio ofNAD(P)H to NAD(P) present in the cytosol of the microbial organismfollowing the disruptions can be an NADH dehydrogenase, a cytochromeoxidase, a glycerol-3-phosphate (G3P) dehydrogenase, aglycerol-3-phosphate (G3P) phosphatase, an alcohol dehydrogenase, apyruvate decarboxylase, an aldehyde dehydrogenase (acid forming), alactate dehydrogenase, a glycerol-3-phosphate dehydrogenase, aglycerol-3-phosphate:quinone oxidoreductase, a malic enzyme and a malatedehydrogenase. In some aspects, the one or more gene disruptions includea deletion of the one or more genes.

In some embodiments, the invention provides a method for producing afatty alcohol, fatty aldehyde or fatty acid using a non-naturallyoccurring microbial organism of the invention that is Crabtree positiveand is in culture medium comprising excess glucose. In such conditions,as described herein, the microbial organism can result in increasing theratio of NAD(P)H to NAD(P) present in the cytosol of the microbialorganism.

In some embodiments, the invention provides a method for producing afatty alcohol, fatty aldehyde or fatty acid using a non-naturallyoccurring microbial organism as described herein having at least oneexogenous nucleic acid encoding an extracellular transporter or anextracellular transport system for a fatty alcohol, fatty aldehyde orfatty acid of the invention.

In some embodiments, the invention provides a method for producing afatty alcohol, fatty aldehyde or fatty acid using a non-naturallyoccurring microbial organism as described herein, wherein one or moreendogenous enzymes involved in: native production of ethanol, glycerol,acetate, formate, lactate, CO₂, fatty acids, or malonyl-CoA by saidmicrobial organism; transfer of pathway intermediates to cellularcompartments other than the cytosol; or native degradation of a MI-FAEcycle intermediate, a MD-FAE cycle intermediate or a termination pathwayintermediate by said microbial organism, has attenuated enzyme activityor expression levels. Accordingly, the endogenous enzyme can be a fattyacid synthase, an acetyl-CoA carboxylase, a biotin:apoenzyme ligase, anacyl carrier protein, a thioesterase, an acyltransferase, an ACPmalonyltransferase, a fatty acid elongase, an acyl-CoA synthetase, anacyl-CoA transferase, an acyl-CoA hydrolase, a pyruvate decarboxylase, alactate dehydrogenase, an alcohol dehydrogenase, an acid-formingaldehyde dehydrogenases, an acetate kinase, a phosphotransacetylase, apyruvate oxidase, a glycerol-3-phosphate dehydrogenase, aglycerol-3-phosphate phosphatase, a mitochondrial pyruvate carrier, aperoxisomal fatty acid transporter, a peroxisomal acyl-CoA transporter,a peroxisomal carnitine/acylcarnitine transferase, an acyl-CoA oxidase,and an acyl-CoA binding protein.

In some embodiments, the invention provides a method for producing afatty alcohol, fatty aldehyde or fatty acid using a non-naturallyoccurring microbial organism as described herein, wherein one or moreendogenous enzymes involved in the oxidation of NAD(P)H or NADH, hasattenuated enzyme activity or expression levels. Accordingly, the one ormore endogenous enzymes can be NADH dehydrogenase, a cytochrome oxidase,a glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate phosphatase,an alcohol dehydrogenase, a pyruvate decarboxylase, an aldehydedehydrogenase (acid forming), a lactate dehydrogenase, aglycerol-3-phosphate dehydrogenase, a glycerol-3-phosphate:quinoneoxidoreductase, a malic enzyme and a malate dehydrogenase.

A fatty alcohol, fatty aldehyde or fatty acid can be harvested orisolated at any time point during the culturing of the microbialorganism, for example, in a continuous and/or near-continuous cultureperiod, as disclosed herein. Generally, the longer the microorganismsare maintained in a continuous and/or near-continuous growth phase, theproportionally greater amount of fatty alcohol, fatty aldehyde or fattyacid can be produced.

Therefore, the invention additionally provides a method for producingfatty alcohol, fatty aldehyde or fatty acid that includes culturing anon-naturally occurring microbial organism having one or more genedisruptions, as disclosed herein. The disruptions can occur in one ormore genes encoding an enzyme that increases production of fattyalcohol, fatty aldehyde or fatty acid, including optionally couplingfatty alcohol, fatty aldehyde or fatty acid production to growth of themicroorganism when the gene disruption reduces or eliminates an activityof the enzyme. For example, the disruptions can confer stablegrowth-coupled production of fatty alcohol, fatty aldehyde or fatty acidonto the non-naturally microbial organism.

In some embodiments, the gene disruption can include a complete genedeletion. In some embodiments other methods to disrupt a gene include,for example, frameshifting by omission or addition of oligonucleotidesor by mutations that render the gene inoperable. One skilled in the artwill recognize the advantages of gene deletions, however, because of thestability it confers to the non-naturally occurring organism fromreverting to a parental phenotype in which the gene disruption has notoccurred. In particular, the gene disruptions are selected from the genesets as disclosed herein.

Once computational predictions are made of gene sets for disruption toincrease production of fatty alcohol, fatty aldehyde or fatty acid, thestrains can be constructed, evolved, and tested. Gene disruptions,including gene deletions, are introduced into host organism by methodswell known in the art. A particularly useful method for gene disruptionis by homologous recombination, as disclosed herein.

The engineered strains can be characterized by measuring the growthrate, the substrate uptake rate, and/or the product/byproduct secretionrate. Cultures can be grown and used as inoculum for a fresh batchculture for which measurements are taken during exponential growth. Thegrowth rate can be determined by measuring optical density using aspectrophotometer (A600). Concentrations of glucose and other organicacid byproducts in the culture supernatant can be determined by wellknown methods such as HPLC, GC-MS or other well known analytical methodssuitable for the analysis of the desired product, as disclosed herein,and used to calculate uptake and secretion rates.

Strains containing gene disruptions can exhibit suboptimal growth ratesuntil their metabolic networks have adjusted to their missingfunctionalities. To assist in this adjustment, the strains can beadaptively evolved. By subjecting the strains to adaptive evolution,cellular growth rate becomes the primary selection pressure and themutant cells are compelled to reallocate their metabolic fluxes in orderto enhance their rates of growth. This reprogramming of metabolism hasbeen recently demonstrated for several E. coli mutants that had beenadaptively evolved on various substrates to reach the growth ratespredicted a priori by an in silico model (Fong and Palsson, Nat. Genet.36:1056-1058 (2004)). The growth improvements brought about by adaptiveevolution can be accompanied by enhanced rates of fatty alcohol, fattyaldehyde or fatty acid production. The strains are generally adaptivelyevolved in replicate, running in parallel, to account for differences inthe evolutionary patterns that can be exhibited by a host organism (Fongand Palsson, Nat. Genet. 36:1056-1058 (2004); Fong et al., J. Bacteriol.185:6400-6408 (2003); Ibarra et al., Nature 420:186-189 (2002)) thatcould potentially result in one strain having superior productionqualities over the others. Evolutions can be run for a period of time,typically 2-6 weeks, depending upon the rate of growth improvementattained. In general, evolutions are stopped once a stable phenotype isobtained.

Following the adaptive evolution process, the new strains arecharacterized again by measuring the growth rate, the substrate uptakerate, and the product/byproduct secretion rate. These results arecompared to the theoretical predictions by plotting actual growth andproduction yields along side the production envelopes from metabolicmodeling. The most successful design/evolution combinations are chosento pursue further, and are characterized in lab-scale batch andcontinuous fermentations. The growth-coupled biochemical productionconcept behind the methods disclosed herein such as OptKnock approachshould also result in the generation of genetically stableoverproducers. Thus, the cultures are maintained in continuous mode foran extended period of time, for example, one month or more, to evaluatelong-term stability. Periodic samples can be taken to ensure that yieldand productivity are maintained.

Adaptive evolution is a powerful technique that can be used to increasegrowth rates of mutant or engineered microbial strains, or of wild-typestrains growing under unnatural environmental conditions. It isespecially useful for strains designed via methods such as OptKnock,which results in growth-coupled product formation. Therefore, evolutiontoward optimal growing strains will indirectly optimize production aswell. Unique strains of E. coli K-12 MG1655 were created through geneknockouts and adaptive evolution. (Fong and Palsson, Nat. Genet.36:1056-1058 (2004)). In this work, all adaptive evolutionary cultureswere maintained in prolonged exponential growth by serial passage ofbatch cultures into fresh medium before the stationary phase wasreached, thus rendering growth rate as the primary selection pressure.Knockout strains were constructed and evolved on minimal mediumsupplemented with different carbon substrates (four for each knockoutstrain). Evolution cultures were carried out in duplicate or triplicate,giving a total of 50 evolution knockout strains. The evolution cultureswere maintained in exponential growth until a stable growth rate wasreached. The computational predictions were accurate (within 10%) atpredicting the post-evolution growth rate of the knockout strains in 38out of the 50 cases examined. Furthermore, a combination of OptKnockdesign with adaptive evolution has led to improved lactic acidproduction strains. (Fong et al., Biotechnol. Bioeng. 91:643-648(2005)). Similar methods can be applied to the strains disclosed hereinand applied to various host strains.

There are a number of developed technologies for carrying out adaptiveevolution. Exemplary methods are disclosed herein. In some embodiments,optimization of a non-naturally occurring organism of the presentinvention includes utilizing adaptive evolution techniques to increasefatty alcohol, fatty aldehyde or fatty acid production and/or stabilityof the producing strain.

Serial culture involves repetitive transfer of a small volume of grownculture to a much larger vessel containing fresh growth medium. When thecultured organisms have grown to saturation in the new vessel, theprocess is repeated. This method has been used to achieve the longestdemonstrations of sustained culture in the literature (Lenski andTravisano, Proc. Natl. Acad. Sci. USA 91:6808-6814 (1994)) inexperiments which clearly demonstrated consistent improvement inreproductive rate over a period of years. Typically, transfer ofcultures is usually performed during exponential phase, so each day thetransfer volume is precisely calculated to maintain exponential growththrough the next 24 hour period. Manual serial dilution is inexpensiveand easy to parallelize.

In continuous culture the growth of cells in a chemostat represents anextreme case of dilution in which a very high fraction of the cellpopulation remains. As a culture grows and becomes saturated, a smallproportion of the grown culture is replaced with fresh media, allowingthe culture to continually grow at close to its maximum population size.Chemostats have been used to demonstrate short periods of rapidimprovement in reproductive rate (Dykhuizen, Methods Enzymol. 613-631(1993)). The potential usefulness of these devices was recognized, buttraditional chemostats were unable to sustain long periods of selectionfor increased reproduction rate, due to the unintended selection ofdilution-resistant (static) variants. These variants are able to resistdilution by adhering to the surface of the chemostat, and by doing so,outcompete less adherent individuals, including those that have higherreproductive rates, thus obviating the intended purpose of the device(Chao and Ramsdell, J. Gen. Microbiol 20:132-138 (1985)). One possibleway to overcome this drawback is the implementation of a device with twogrowth chambers, which periodically undergo transient phases ofsterilization, as described previously (Marliere and Mutzel, U.S. Pat.No. 6,686,194).

Evolugator™ is a continuous culture device developed by Evolugate, LLC(Gainesville, Fla.) and exhibits significant time and effort savingsover traditional evolution techniques (de Crecy et al., Appl. Microbiol.Biotechnol. 77:489-496 (2007)). The cells are maintained in prolongedexponential growth by the serial passage of batch cultures into freshmedium before the stationary phase is attained. By automating opticaldensity measurement and liquid handling, the Evolugator™ can performserial transfer at high rates using large culture volumes, thusapproaching the efficiency of a chemostat in evolution of cell fitness.For example, a mutant of Acinetobacter sp ADP1 deficient in a componentof the translation apparatus, and having severely hampered growth, wasevolved in 200 generations to 80% of the wild-type growth rate. However,in contrast to the chemostat which maintains cells in a single vessel,the machine operates by moving from one “reactor” to the next insubdivided regions of a spool of tubing, thus eliminating any selectionfor wall-growth. The transfer volume is adjustable, and normally set toabout 50%. A drawback to this device is that it is large and costly,thus running large numbers of evolutions in parallel is not practical.Furthermore, gas addition is not well regulated, and strict anaerobicconditions are not maintained with the current device configuration.Nevertheless, this is an alternative method to adaptively evolve aproduction strain.

As disclosed herein, a nucleic acid encoding a desired activity of afatty alcohol, fatty aldehyde or fatty acid pathway can be introducedinto a host organism. In some cases, it can be desirable to modify anactivity of a fatty alcohol, fatty aldehyde or fatty acid pathway enzymeor protein to increase production of fatty alcohol, fatty aldehyde orfatty acid. For example, known mutations that increase the activity of aprotein or enzyme can be introduced into an encoding nucleic acidmolecule. Additionally, optimization methods can be applied to increasethe activity of an enzyme or protein and/or decrease an inhibitoryactivity, for example, decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Otten and Quax. Biomol. Eng 22:1-9 (2005).; and Sen et al., ApplBiochem. Biotechnol 143:212-223 (2007)) to be effective at creatingdiverse variant libraries, and these methods have been successfullyapplied to the improvement of a wide range of properties across manyenzyme classes. Enzyme characteristics that have been improved and/oraltered by directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates;temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (K_(i)), to remove inhibitionby products, substrates, or key intermediates; activity (kcat), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

Described below in more detail are exemplary methods that have beendeveloped for the mutagenesis and diversification of genes to targetdesired properties of specific enzymes. Such methods are well known tothose skilled in the art. Any of these can be used to alter and/oroptimize the activity of a fatty alcohol, fatty aldehyde or fatty acidpathway enzyme or protein.

EpPCR (Pritchard et al., J Theor. Biol. 234:497-509 (2005)) introducesrandom point mutations by reducing the fidelity of DNA polymerase in PCRreactions by the addition of Mn²⁺ ions, by biasing dNTP concentrations,or by other conditional variations. The five step cloning process toconfine the mutagenesis to the target gene of interest involves: 1)error-prone PCR amplification of the gene of interest; 2) restrictionenzyme digestion; 3) gel purification of the desired DNA fragment; 4)ligation into a vector; 5) transformation of the gene variants into asuitable host and screening of the library for improved performance.This method can generate multiple mutations in a single genesimultaneously, which can be useful to screen a larger number ofpotential variants having a desired activity. A high number of mutantscan be generated by EpPCR, so a high-throughput screening assay or aselection method, for example, using robotics, is useful to identifythose with desirable characteristics.

Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., NucleicAcids Res. 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497(2006)) has many of the same elements as epPCR except a whole circularplasmid is used as the template and random timers with exonucleaseresistant thiophosphate linkages on the last 2 nucleotides are used toamplify the plasmid followed by transformation into cells in which theplasmid is re-circularized at tandem repeats. Adjusting the Mn²⁺concentration can vary the mutation rate somewhat. This technique uses asimple error-prone, single-step method to create a full copy of theplasmid with 3-4 mutations/kbp. No restriction enzyme digestion orspecific primers are required. Additionally, this method is typicallyavailable as a commercially available kit.

DNA or Family Shuffling (Stemmer, Proc Natl Acad Sci USA 91:10747-10751(1994)); and Stemmer, Nature 370:389-391 (1994)) typically involvesdigestion of two or more variant genes with nucleases such as Dnase I orEndoV to generate a pool of random fragments that are reassembled bycycles of annealing and extension in the presence of DNA polymerase tocreate a library of chimeric genes. Fragments prime each other andrecombination occurs when one copy primes another copy (templateswitch). This method can be used with >1 kbp DNA sequences. In additionto mutational recombinants created by fragment reassembly, this methodintroduces point mutations in the extension steps at a rate similar toerror-prone PCR. The method can be used to remove deleterious, randomand neutral mutations.

Staggered Extension (StEP) (Zhao et al., Nat. Biotechnol. 16:258-261(1998)) entails template priming followed by repeated cycles of 2 stepPCR with denaturation and very short duration of annealing/extension (asshort as 5 sec). Growing fragments anneal to different templates andextend further, which is repeated until full-length sequences are made.Template switching means most resulting fragments have multiple parents.Combinations of low-fidelity polymerases (Taq and Mutazyme) reduceerror-prone biases because of opposite mutational spectra.

In Random Priming Recombination (RPR) random sequence primers are usedto generate many short DNA fragments complementary to different segmentsof the template (Shao et al., Nucleic Acids Res 26:681-683 (1998)). Basemisincorporation and mispriming via epPCR give point mutations. ShortDNA fragments prime one another based on homology and are recombined andreassembled into full-length by repeated thermocycling. Removal oftemplates prior to this step assures low parental recombinants. Thismethod, like most others, can be performed over multiple iterations toevolve distinct properties. This technology avoids sequence bias, isindependent of gene length, and requires very little parent DNA for theapplication.

In Heteroduplex Recombination linearized plasmid DNA is used to formheteroduplexes that are repaired by mismatch repair (Volkov et al,Nucleic Acids Res. 27:e18 (1999); and Volkov et al., Methods Enzymol.328:456-463 (2000)). The mismatch repair step is at least somewhatmutagenic. Heteroduplexes transform more efficiently than linearhomoduplexes. This method is suitable for large genes and whole operons.

Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al.,Nat. Biotechnol. 19:354-359 (2001)) employs Dnase I fragmentation andsize fractionation of single stranded DNA (ssDNA). Homologous fragmentsare hybridized in the absence of polymerase to a complementary ssDNAscaffold. Any overlapping unhybridized fragment ends are trimmed down byan exonuclease. Gaps between fragments are filled in and then ligated togive a pool of full-length diverse strands hybridized to the scaffold,which contains U to preclude amplification. The scaffold then isdestroyed and is replaced by a new strand complementary to the diversestrand by PCR amplification. The method involves one strand (scaffold)that is from only one parent while the priming fragments derive fromother genes, and the parent scaffold is selected against. Thus, noreannealing with parental fragments occurs. Overlapping fragments aretrimmed with an exonuclease. Otherwise, this is conceptually similar toDNA shuffling and StEP. Therefore, there should be no siblings, fewinactives, and no unshuffled parentals. This technique has advantages inthat few or no parental genes are created and many more crossovers canresult relative to standard DNA shuffling.

Recombined Extension on Truncated templates (RETT) entails templateswitching of unidirectionally growing strands from primers in thepresence of unidirectional ssDNA fragments used as a pool of templates(Lee et al., J. Molec. Catalysis 26:119-129 (2003)). No DNAendonucleases are used. Unidirectional ssDNA is made by DNA polymerasewith random primers or serial deletion with exonuclease. UnidirectionalssDNA are only templates and not primers. Random priming andexonucleases do not introduce sequence bias as true of enzymaticcleavage of DNA shuffling/RACHITT. RETT can be easier to optimize thanStEP because it uses normal PCR conditions instead of very shortextensions. Recombination occurs as a component of the PCR steps, thatis, no direct shuffling. This method can also be more random than StEPdue to the absence of pauses.

In Degenerate Oligonucleotide Gene Shuffling (DOGS) degenerate primersare used to control recombination between molecules; (Bergquist andGibbs, Methods Mol. Biol 352:191-204 (2007); Bergquist et al., Biomol.Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)) this can beused to control the tendency of other methods such as DNA shuffling toregenerate parental genes. This method can be combined with randommutagenesis (epPCR) of selected gene segments. This can be a good methodto block the reformation of parental sequences. No endonucleases areneeded. By adjusting input concentrations of segments made, one can biastowards a desired backbone. This method allows DNA shuffling fromunrelated parents without restriction enzyme digests and allows a choiceof random mutagenesis methods.

Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY)creates a combinatorial library with 1 base pair deletions of a gene orgene fragment of interest (Ostermeier et al., Proc. Natl. Acad. Sci. USA96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol.17:1205-1209 (1999)). Truncations are introduced in opposite directionon pieces of 2 different genes. These are ligated together and thefusions are cloned. This technique does not require homology between the2 parental genes. When ITCHY is combined with DNA shuffling, the systemis called SCRATCHY (see below). A major advantage of both is no need forhomology between parental genes; for example, functional fusions betweenan E. coli and a human gene were created via ITCHY. When ITCHY librariesare made, all possible crossovers are captured.

Thio-Incremental Truncation for the Creation of Hybrid Enzymes(THIO-ITCHY) is similar to ITCHY except that phosphothioate dNTPs areused to generate truncations (Lutz et al., Nucleic Acids Res 29:E16(2001)). Relative to ITCHY, THIO-ITCHY can be easier to optimize,provide more reproducibility, and adjustability.

SCRATCHY combines two methods for recombining genes, ITCHY and DNAshuffling (Lutz et al., Proc. Natl. Acad. Sci. USA 98:11248-11253(2001)). SCRATCHY combines the best features of ITCHY and DNA shuffling.First, ITCHY is used to create a comprehensive set of fusions betweenfragments of genes in a DNA homology-independent fashion. Thisartificial family is then subjected to a DNA-shuffling step to augmentthe number of crossovers. Computational predictions can be used inoptimization. SCRATCHY is more effective than DNA shuffling whensequence identity is below 80%.

In Random Drift Mutagenesis (RNDM) mutations are made via epPCR followedby screening/selection for those retaining usable activity (Bergquist etal., Biomol. Eng. 22:63-72 (2005)). Then, these are used in DOGS togenerate recombinants with fusions between multiple active mutants orbetween active mutants and some other desirable parent. Designed topromote isolation of neutral mutations; its purpose is to screen forretained catalytic activity whether or not this activity is higher orlower than in the original gene. RNDM is usable in high throughputassays when screening is capable of detecting activity above background.RNDM has been used as a front end to DOGS in generating diversity. Thetechnique imposes a requirement for activity prior to shuffling or othersubsequent steps; neutral drift libraries are indicated to result inhigher/quicker improvements in activity from smaller libraries. Thoughpublished using epPCR, this could be applied to other large-scalemutagenesis methods.

Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis methodthat: 1) generates a pool of random length fragments using randomincorporation of a phosphothioate nucleotide and cleavage; this pool isused as a template to 2) extend in the presence of “universal” basessuch as inosine; 3) replication of an inosine-containing complementgives random base incorporation and, consequently, mutagenesis (Wong etal., Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res.32:e26 (2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)).Using this technique it can be possible to generate a large library ofmutants within 2 to 3 days using simple methods. This technique isnon-directed in comparison to the mutational bias of DNA polymerases.Differences in this approach makes this technique complementary (or analternative) to epPCR.

In Synthetic Shuffling, overlapping oligonucleotides are designed toencode “all genetic diversity in targets” and allow a very highdiversity for the shuffled progeny (Ness et al., Nat. Biotechnol.20:1251-1255 (2002)). In this technique, one can design the fragments tobe shuffled. This aids in increasing the resulting diversity of theprogeny. One can design sequence/codon biases to make more distantlyrelated sequences recombine at rates approaching those observed withmore closely related sequences. Additionally, the technique does notrequire physically possessing the template genes.

Nucleotide Exchange and Excision Technology NexT exploits a combinationof dUTP incorporation followed by treatment with uracil DNA glycosylaseand then piperidine to perform endpoint DNA fragmentation (Muller etal., Nucleic Acids Res. 33:e117 (2005)). The gene is reassembled usinginternal PCR primer extension with proofreading polymerase. The sizesfor shuffling are directly controllable using varying dUPT:dTTP ratios.This is an end point reaction using simple methods for uracilincorporation and cleavage. Other nucleotide analogs, such as8-oxo-guanine, can be used with this method. Additionally, the techniqueworks well with very short fragments (86 bp) and has a low error rate.The chemical cleavage of DNA used in this technique results in very fewunshuffled clones.

In Sequence Homology-Independent Protein Recombination (SHIPREC), alinker is used to facilitate fusion between two distantly related orunrelated genes. Nuclease treatment is used to generate a range ofchimeras between the two genes. These fusions result in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)). This produces a limited type of shuffling and a separateprocess is required for mutagenesis. In addition, since no homology isneeded, this technique can create a library of chimeras with varyingfractions of each of the two unrelated parent genes. SHIPREC was testedwith a heme-binding domain of a bacterial CP450 fused to N-terminalregions of a mammalian CP450; this produced mammalian activity in a moresoluble enzyme.

In Gene Site Saturation Mutagenesis™ (GSSM™) the starting materials area supercoiled dsDNA plasmid containing an insert and two primers whichare degenerate at the desired site of mutations (Kretz et al., MethodsEnzymol. 388:3-11 (2004)). Primers carrying the mutation of interest,anneal to the same sequence on opposite strands of DNA. The mutation istypically in the middle of the primer and flanked on each side byapproximately 20 nucleotides of correct sequence. The sequence in theprimer is NNN or NNK (coding) and MNN (noncoding) (N=all 4, K=G, T, M=A,C). After extension, DpnI is used to digest dam-methylated DNA toeliminate the wild-type template. This technique explores all possibleamino acid substitutions at a given locus (that is, one codon). Thetechnique facilitates the generation of all possible replacements at asingle-site with no nonsense codons and results in equal to near-equalrepresentation of most possible alleles. This technique does not requireprior knowledge of the structure, mechanism, or domains of the targetenzyme. If followed by shuffling or Gene Reassembly, this technologycreates a diverse library of recombinants containing all possiblecombinations of single-site up-mutations. The usefulness of thistechnology combination has been demonstrated for the successfulevolution of over 50 different enzymes, and also for more than oneproperty in a given enzyme.

Combinatorial Cassette Mutagenesis (CCM) involves the use of shortoligonucleotide cassettes to replace limited regions with a large numberof possible amino acid sequence alterations (Reidhaar-Olson et al.Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science241:53-57 (1988)). Simultaneous substitutions at two or three sites arepossible using this technique. Additionally, the method tests a largemultiplicity of possible sequence changes at a limited range of sites.This technique has been used to explore the information content of thelambda repressor DNA-binding domain.

Combinatorial Multiple Cassette Mutagenesis (CMCM) is essentiallysimilar to CCM except it is employed as part of a larger program: 1) useof epPCR at high mutation rate to 2) identify hot spots and hot regionsand then 3) extension by CMCM to cover a defined region of proteinsequence space (Reetz et al., Angew. Chem. Int. Ed Engl. 40:3589-3591(2001)). As with CCM, this method can test virtually all possiblealterations over a target region. If used along with methods to createrandom mutations and shuffled genes, it provides an excellent means ofgenerating diverse, shuffled proteins. This approach was successful inincreasing, by 51-fold, the enantioselectivity of an enzyme.

In the Mutator Strains technique, conditional ts mutator plasmids allowincreases of 20 to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)). This technology is based on a plasmid-derivedmutD5 gene, which encodes a mutant subunit of DNA polymerase III. Thissubunit binds to endogenous DNA polymerase III and compromises theproofreading ability of polymerase III in any strain that harbors theplasmid. A broad-spectrum of base substitutions and frameshift mutationsoccur. In order for effective use, the mutator plasmid should be removedonce the desired phenotype is achieved; this is accomplished through atemperature sensitive (ts) origin of replication, which allows forplasmid curing at 41° C. It should be noted that mutator strains havebeen explored for quite some time (see Low et al., J. Mol. Biol.260:359-3680 (1996)). In this technique, very high spontaneous mutationrates are observed. The conditional property minimizes non-desiredbackground mutations. This technology could be combined with adaptiveevolution to enhance mutagenesis rates and more rapidly achieve desiredphenotypes.

Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis methodthat assesses and optimizes combinatorial mutations of selected aminoacids (Rajpal et al., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)).Rather than saturating each site with all possible amino acid changes, aset of nine is chosen to cover the range of amino acid R-groupchemistry. Fewer changes per site allows multiple sites to be subjectedto this type of mutagenesis. A >800-fold increase in binding affinityfor an antibody from low nanomolar to picomolar has been achievedthrough this method. This is a rational approach to minimize the numberof random combinations and can increase the ability to find improvedtraits by greatly decreasing the numbers of clones to be screened. Thishas been applied to antibody engineering, specifically to increase thebinding affinity and/or reduce dissociation. The technique can becombined with either screens or selections.

Gene Reassembly is a DNA shuffling method that can be applied tomultiple genes at one time or to create a large library of chimeras(multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™)Technology supplied by Verenium Corporation). Typically this technologyis used in combination with ultra-high-throughput screening to query therepresented sequence space for desired improvements. This techniqueallows multiple gene recombination independent of homology. The exactnumber and position of cross-over events can be pre-determined usingfragments designed via bioinformatic analysis. This technology leads toa very high level of diversity with virtually no parental genereformation and a low level of inactive genes. Combined with GSSM™, alarge range of mutations can be tested for improved activity. The methodallows “blending” and “fine tuning” of DNA shuffling, for example, codonusage can be optimized.

In Silico Protein Design Automation (PDA) is an optimization algorithmthat anchors the structurally defined protein backbone possessing aparticular fold, and searches sequence space for amino acidsubstitutions that can stabilize the fold and overall protein energetics(Hayes et al., Proc. Natl. Acad. Sci. USA 99:15926-15931 (2002)). Thistechnology uses in silico structure-based entropy predictions in orderto search for structural tolerance toward protein amino acid variations.Statistical mechanics is applied to calculate coupling interactions ateach position. Structural tolerance toward amino acid substitution is ameasure of coupling. Ultimately, this technology is designed to yielddesired modifications of protein properties while maintaining theintegrity of structural characteristics. The method computationallyassesses and allows filtering of a very large number of possiblesequence variants (10⁵⁰). The choice of sequence variants to test isrelated to predictions based on the most favorable thermodynamics.Ostensibly only stability or properties that are linked to stability canbe effectively addressed with this technology. The method has beensuccessfully used in some therapeutic proteins, especially inengineering immunoglobulins. In silico predictions avoid testingextraordinarily large numbers of potential variants. Predictions basedon existing three-dimensional structures are more likely to succeed thanpredictions based on hypothetical structures. This technology canreadily predict and allow targeted screening of multiple simultaneousmutations, something not possible with purely experimental technologiesdue to exponential increases in numbers.

Iterative Saturation Mutagenesis (ISM) involves: 1) using knowledge ofstructure/function to choose a likely site for enzyme improvement; 2)performing saturation mutagenesis at chosen site using a mutagenesismethod such as Stratagene QuikChange (Stratagene; San Diego Calif.); 3)screening/selecting for desired properties; and 4) using improvedclone(s), start over at another site and continue repeating until adesired activity is achieved (Reetz et al., Nat. Protoc. 2:891-903(2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751(2006)). This is a proven methodology, which assures all possiblereplacements at a given position are made for screening/selection.

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Production of Fatty Alcohols and Fatty Aldehydes by MI-FAECycle, MD-FAE Cycle and Acyl-CoA Termination Pathways

Encoding nucleic acids and species that can be used as sources forconferring fatty alcohol and fatty aldehyde production capability onto ahost microbial organism are exemplified further below.

Multienzyme Complexes

In one exemplary embodiment, the genes fadA and fadB encode amultienzyme complex that exhibits three constituent activities of themalonyl-CoA independent FAS pathway, namely, ketoacyl-CoA thiolase,3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydratase activities(Nakahigashi, K. and H. Inokuchi, Nucleic Acids Research 18:4937 (1990);Yang et al., Journal of Bacteriology 173:7405-7406 (1991); Yang et al,Journal of Biological Chemistry 265:10424-10429 (1990); Yang et al.,Biochemistry 30:6788-6795 (1990)). The fadI and fadJ genes encodesimilar activities which can substitute for the above malonyl-CoAindependent FAS conferring genes fadA and fadB. The acyl-Coadehydrogenase of E. coli is encoded by fadE (Campbell et al, J Bacteriol184: 3759-64)). This enzyme catalyzes the rate-limiting step ofbeta-oxidation (O'Brien et al, J Bacteriol 132:532-40 (1977)). Thenucleic acid sequences for each of the above fad genes are well known inthe art and can be accessed in the public databases such as Genbankusing the following accession numbers.

Protein GenBank ID GI Number Organism fadA YP_026272.1 49176430Escherichia coli fadB NP_418288.1 16131692 Escherichia coli fadINP_416844.1 16130275 Escherichia coli fadJ NP_416843.1 16130274Escherichia coli fadR NP_415705.1 16129150 Escherichia coli fadEAAC73325.2 87081702 Escherichia coli

Step A. Thiolase

Thiolase enzymes, also know as beta-keto thiolase, acyl-CoAC-acetyltransferase, acyl-CoA:acetyl-CoA C-acyltransferase,3-oxoacyl-CoA thiolase, 3-ketoacyl-CoA thiolase, beta-ketoacyl-CoAthiolase, and acyl-CoA thiolase, that are suitable for fatty alcohol,fatty aldehyde or fatty acid production are described herein (FIGS. 1Aand 6A). Exemplary acetoacetyl-CoA thiolase enzymes include the geneproducts of atoB and homolog yqeF from E. coli (Martin et al., Nat.Biotechnol 21:796-802 (2003)), thlA and thlB from C. acetobutylicum(Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007); Winzer etal., J. Mol. Microbiol Biotechnol 2:531-541 (2000)), and ERG10 from S.cerevisiae (Hiser et al., J. Biol. Chem. 269:31383-31389 (1994)). Adegradative thiolase of S. cerevisiae is encoded by POT1. Anothercandidate thiolase is the phaA gene product of R. eutropha (Jenkins etal, Journal of Bacteriology 169:42-52 (1987)). The acetoacetyl-CoAthiolase from Zoogloea ramigera is irreversible in the biosyntheticdirection and a crystal structure is available (Merilainen et al,Biochem 48: 11011-25 (2009)). Accession numbers for these thiolases andhomologs are included in the table below.

Protein GenBank ID GI Number Organism atoB NP_416728 16130161Escherichia coli yqeF NP_417321.2 90111494 Escherichia coli thlANP_349476.1 15896127 Clostridium acetobutylicum thlB NP_149242.115004782 Clostridium acetobutylicum ERG10 NP_015297 6325229Saccharomyces cerevisiae POT1 NP_012106.1 6322031 Saccharomycescerevisiae phaA YP_725941 113867452 Ralstonia eutropha phbA P07097.4135759 Zoogloea ramigera h16_A1713 YP_726205.1 113867716 Ralstoniaeutropha pcaF YP_728366.1 116694155 Ralstonia eutropha h16_B1369YP_840888.1 116695312 Ralstonia eutropha h16_A0170 YP_724690.1 113866201Ralstonia eutropha h16_A0462 YP_724980.1 113866491 Ralstonia eutrophah16_A1528 YP_726028.1 113867539 Ralstonia eutropha h16_B0381 YP_728545.1116694334 Ralstonia eutropha h16_B0662 YP_728824.1 116694613 Ralstoniaeutropha h16_B0759 YP_728921.1 116694710 Ralstonia eutropha h16_B0668YP_728830.1 116694619 Ralstonia eutropha h16_A1720 YP_726212.1 113867723Ralstonia eutropha h16_A1887 YP_726356.1 113867867 Ralstonia eutrophabktB YP_002005382.1 194289475 Cupriavidus taiwanensis Rmet_1362YP_583514.1 94310304 Ralstonia metallidurans Bphy_0975 YP_001857210.1186475740 Burkholderia phymatum

Many thiolase enzymes catalyze the formation of longer-chain acyl-CoAproducts. Exemplary thiolases include, for example, 3-oxoadipyl-CoAthiolase (EC 2.3.1.174) and acyl-CoA thiolase (EC 2.3.1.16).3-Oxoadipyl-CoA thiolase converts succinyl-CoA and acetyl-CoA to3-oxoadipyl-CoA, and is a key enzyme of the beta-ketoadipate pathway foraromatic compound degradation. The enzyme is widespread in soil bacteriaand fungi including Pseudomonas putida (Harwood et al., J Bacteriol.176:6479-6488 (1994)) and Acinetobacter calcoaceticus (Doten et al., JBacteriol. 169:3168-3174 (1987)). The gene products encoded by pcaF inPseudomonas strain B13 (Kaschabek et al., J Bacteriol. 184:207-215(2002)), phaD in Pseudomonas putida U (Olivera et al., Proc. Natl. Acad.Sci U.S.A 95:6419-6424 (1998)), paaE in Pseudomonas fluorescens ST (Diet al., Arch. Microbiol 188:117-125 (2007)), and paaJ from E. coli(Nogales et al., Microbiology 153:357-365 (2007)) also catalyze thistransformation. Several beta-ketothiolases exhibit significant andselective activities in the oxoadipyl-CoA forming direction includingbkt from Pseudomonas putida, pcaF and bkt from Pseudomonas aeruginosaPAO1, bkt from Burkholderia ambifaria AMMD, paaJ from E. coli, and phaDfrom P. putida. Two gene products of Ralstonia eutropha (formerly knownas Alcaligenes eutrophus), encoded by genes bktB and bktC, catalyze theformation of 3-oxopimeloyl-CoA (Slater et al., J. Bacteriol.180:1979-1987 (1998); Haywood et al., FEMS Microbiology Letters 52:91-96(1988)). The sequence of the BktB protein is known; however, thesequence of the BktC protein has not been reported. BktB is also activeon substrates of length C6 and C8 (Machado et al, Met Eng in press(2012)). The pim operon of Rhodopseudomonas palustris also encodes abeta-ketothiolase, encoded by pimB, predicted to catalyze thistransformation in the degradative direction during benzoyl-CoAdegradation (Harrison et al., Microbiology 151:727-736 (2005)). Abeta-ketothiolase enzyme candidate in S. aciditrophicus was identifiedby sequence homology to bktB (43% identity, evalue=1e-93).

GenBank Gene name GI# Accession # Organism paaJ 16129358 NP_415915.1Escherichia coli pcaF 17736947 AAL02407 Pseudomonas knackmussii (B13)phaD 3253200 AAC24332.1 Pseudomonas putida pcaF 506695 AAA85138.1Pseudomonas putida pcaF 141777 AAC37148.1 Acinetobacter calcoaceticuspaaE 106636097 ABF82237.1 Pseudomonas fluorescens bkt 115360515YP_777652.1 Burkholderia ambifaria AMMD bkt 9949744 AAG06977.1Pseudomonas aeruginosa PAO1 pcaF 9946065 AAG03617.1 Pseudomonasaeruginosa PAO1 bktB YP_725948 11386745 Ralstonia eutropha pimB CAE2915639650633 Rhodopseudomonas palustris syn_02642 YP_462685.1 85860483Syntrophus aciditrophicus

Acyl-CoA thiolase (EC 2.3.1.16) enzymes involved in the beta-oxidationcycle of fatty acid degradation exhibit activity on a broad range ofacyl-CoA substrates of varying chain length. Exemplary acyl-CoAthiolases are found in Arabidopsis thaliana (Cruz et al, Plant Physiol135:85-94 (2004)), Homo sapiens (Mannaerts et al, Cell Biochem Biphys32:73-87 (2000)), Helianthus annuus (Schiedel et al, Prot Expr Purif33:25-33 (2004)). The chain length specificity of thiolase enzymes canbe assayed by methods well known in the art (Wrensford et al, AnalBiochem 192:49-54 (1991)). A peroxisomal thiolase found in rat livercatalyze the acetyl-CoA dependent formation of longer chain acyl-CoAproducts from octanoyl-CoA (Horie et al, Arch Biochem Biophys 274: 64-73(1989); Hijikata et al, J Biol Chem 265, 4600-4606 (1990)).

Protein GenBank ID GI Number Organism AY308827.1: AAQ77242.1 34597334Helianthus annuus 1 . . . 1350 KAT2 Q56WD9.2 73919871 Arabidopsisthaliana KAT1 Q8LF48.2 73919870 Arabidopsis thaliana KAT5 Q570C8.273919872 Arabidopsis thaliana ACAA1 P09110.2 135751 Homo sapiens LCTHIOAAF04612.1 6165556 Sus scrofa Acaa1a NP_036621.1 6978429 Rattusnorvegicus Acaa1b NP_001035108.1 90968642 Rattus norvegicus Acaa2NP_569117.1 18426866 Rattus norvegicus

Acetoacetyl-CoA can also be synthesized from acetyl-CoA and malonyl-CoAby acetoacetyl-CoA synthase (EC 2.3.1.194). This enzyme (FhsA) has beencharacterized in the soil bacterium Streptomyces sp. CL190 where itparticipates in mevalonate biosynthesis (Okamura et al, PNAS USA107:11265-70 (2010)). As this enzyme catalyzes an essentiallyirreversible reaction, it is particularly useful for metabolicengineering applications for overproducing metabolites, fuels orchemicals derived from acetoacetyl-CoA such as long chain alcohols.Other acetoacetyl-CoA synthase genes can be identified by sequencehomology to fhsA. Acyl-CoA synthase enzymes such as fhsA and homologscan be engineered or evolved to accept longer acyl-CoA substrates bymethods known in the art.

Protein GenBank ID GI Number Organism fhsA BAJ83474.1 325302227Streptomyces sp CL190 AB183750.1: BAD86806.1 57753876 Streptomyces 11991. . . 12971 sp. KO-3988 epzT ADQ43379.1 312190954 Streptomycescinnamonensis ppzT CAX48662.1 238623523 Streptomyces anulatus O3I_22085ZP_09840373.1 378817444 Nocardia brasiliensis

Chain length selectivity of selected thiolase enzymes described above issummarized in the table below.

Chain length Gene Organism C4 atoB Escherichia coli C6 phaD Pseudomonasputida C6-C8 bktB Ralstonia eutropha C10-C16 Acaa1a Rattus norvegicus

Step B. 3-Oxoacyl-CoA Reductase

3-Oxoacyl-CoA reductases (also known as 3-hydroxyacyl-CoAdehydrogenases, 3-ketoacyl-CoA reductases, beta-ketoacyl-CoA reductases,beta-hydroxyacyl-CoA dehydrogenases, hydroxyacyl-CoA dehydrogenases, andketoacyl-CoA reductases) catalyze the reduction of 3-oxoacyl-CoAsubstrates to 3-hydroxyacyl-CoA products (FIG. 1B and FIG. 6B). Theseenzymes are often involved in fatty acid beta-oxidation and aromaticdegradation pathways. For example, subunits of two fatty acid oxidationcomplexes in E. coli, encoded by fadB and fadJ, function as3-hydroxyacyl-CoA dehydrogenases (Binstock et al., Methods Enzymol. 71Pt C:403-411 (1981)). Knocking out a negative regulator encoded by fadRcan be utilized to activate the fadB gene product (Sato et al., JBiosci. Bioeng 103:38-44 (2007)). Another 3-hydroxyacyl-CoAdehydrogenase from E. coli is paaH (Ismail et al., European Journal ofBiochemistry 270:3047-3054 (2003)). Additional 3-oxoacyl-CoA enzymesinclude the gene products of phaC in Pseudomonas putida (Olivera et al.,Proc. Natl. Acad. Sci U.S.A 95:6419-6424 (1998)) and paaC in Pseudomonasfluorescens (Di et al., 188:117-125 (2007)). These enzymes catalyze thereversible oxidation of 3-hydroxyadipyl-CoA to 3-oxoadipyl-CoA duringthe catabolism of phenylacetate or styrene. Other suitable enzymecandidates include AAO72312.1 from E. gracilis (Winkler et al., PlantPhysiology 131:753-762 (2003)) and paaC from Pseudomonas putida (Oliveraet al., PNAS USA 95:6419-6424 (1998)). Enzymes catalyzing the reductionof acetoacetyl-CoA to 3-hydroxybutyryl-CoA include hbd of Clostridiumacetobutylicum (Youngleson et al., J Bacteriol. 171:6800-6807 (1989)),phbB from Zoogloea ramigera (Ploux et al., Eur. J Biochem. 174:177-182(1988)), phaB from Rhodobacter sphaeroides (Alber et al., Mol. Microbiol61:297-309 (2006)) and paaH1 of Ralstonia eutropha (Machado et al, MetEng, In Press (2012)). The Z. ramigera enzyme is NADPH-dependent andalso accepts 3-oxopropionyl-CoA as a substrate (Ploux et al., Eur. JBiochem. 174:177-182 (1988)). Additional genes include phaB inParacoccus denitrificans, Hbd1 (C-terminal domain) and Hbd2 (N-terminaldomain) in Clostridium kluyveri (Hillmer and Gottschalk, Biochim.Biophys. Acta 3334:12-23 (1974)) and HSD17B10 in Bos taurus (Wakil etal., J Biol. Chem. 207:631-638 (1954)). The enzyme from Paracoccusdenitrificans has been functionally expressed and characterized in E.coli (Yabutani et al., FEMS Microbiol Lett. 133:85-90 (1995)). A numberof similar enzymes have been found in other species of Clostridia and inMetallosphaera sedula (Berg et al., Science. 318:1782-1786 (2007)). Theenzyme from Candida tropicalis is a component of the peroxisomal fattyacid beta-oxidation multifunctional enzyme type 2 (MFE-2). Thedehydrogenase B domain of this protein is catalytically active onacetoacetyl-CoA. The domain has been functionally expressed in E. coli,a crystal structure is available, and the catalytic mechanism iswell-understood (Ylianttila et al., Biochem Biophys Res Commun 324:25-30(2004); Ylianttila et al., J Mol Biol 358:1286-1295 (2006)).3-Hydroxyacyl-CoA dehydrogenases that accept longer acyl-CoA substrates(eg. EC 1.1.1.35) are typically involved in beta-oxidation. An exampleis HSD17B10 in Bos taurus (Wakil et al., J Biol. Chem. 207:631-638(1954)). The pig liver enzyme is preferentially active on short andmedium chain acyl-CoA substrates whereas the heart enzyme is lessselective (He et al, Biochim Biophys Acta 1392:119-26 (1998)). The S.cerevisiae enzyme FOX2 is active in beta-degradation pathways and alsohas enoyl-CoA hydratase activity (Hiltunen et al, J Biol Chem 267:6646-6653 (1992)).

Protein Genbank ID GI number Organism fadB P21177.2 119811 Escherichiacoli fadJ P77399.1 3334437 Escherichia coli paaH NP_415913.1 16129356Escherichia coli Hbd2 EDK34807.1 146348271 Clostridium kluyveri Hbd1EDK32512.1 146345976 Clostridium kluyveri phaC NP_745425.1 26990000Pseudomonas putida paaC ABF82235.1 106636095 Pseudomonas fluorescensHSD17B10 O02691.3 3183024 Bos taurus phbB P23238.1 130017 Zoogloearamigera phaB YP_353825.1 77464321 Rhodobacter sphaeroides paaH1CAJ91433.1 113525088 Ralstonia eutropha phaB BAA08358 675524 Paracoccusdenitriftcans Hbd NP_349314.1 15895965 Clostridium acetobutylicum HbdAAM14586.1 20162442 Clostridium beijerinckii Msed_1423 YP_001191505146304189 Metallosphaera sedula Msed_0399 YP_001190500 146303184Metallosphaera sedula Msed_0389 YP_001190490 146303174 Metallosphaerasedula Msed_1993 YP_001192057 146304741 Metallosphaera sedula Fox2Q02207 399508 Candida tropicalis HSD17B10 O02691.3 3183024 Bos taurusHADH NP_999496.1 47523722 Bos taurus 3HCDH AAO72312.1 29293591 Euglenagracilis FOX2 NP_012934.1 6322861 Saccharomyces cerevisiae

Chain length specificity of selected hydroxyacyl-CoA dehydrogenaseenzymes is shown below. Directed evolution can enhance selectivity ofenzymes for longer-chain substrates. For example, Machado and coworkersdeveloped a selection platform for directed evolution of chainelongation enzymes that favor longer acyl-CoA substrates. This groupevolved paaH1 of Ralstonia eutropha for improved activity on3-oxo-hexanoyl-CoA (Machado et al, Met Eng, In Press (2012)).

Chain length Gene Organism C4 hbd Clostridium acetobutylicum C5 phbBZoogloea ramigera C4-C6 paaH1 Ralstonia eutropha C4-C10 HADH Sus scrofaC4-C18 fadB Escherichia coliStep C. 3-Hydroxyacyl-CoA Dehydratase 3-Hydroxyacyl-CoA dehydratases(eg. EC 4.2.1.17, also known as enoyl-CoA hydratases) catalyze thedehydration of a range of 3-hydroxyacyl-CoA substrates (Roberts et al.,Arch. Microbiol 117:99-108 (1978); Agnihotri et al., Bioorg. Med. Chem.11:9-20 (2003); Conrad et al., J Bacteriol. 118:103-111 (1974)) and canbe used in the conversion of 3-hydroxyacyl-CoA to enoyl-CoA (FIGS. 1Cand 6C). The ech gene product of Pseudomonas putida catalyzes theconversion of 3-hydroxybutyryl-CoA to crotonyl-CoA (Roberts et al.,Arch. Microbiol 117:99-108 (1978)). This transformation is alsocatalyzed by the crt gene product of Clostridium acetobutylicum, thecrt1 gene product of C. kluyveri, and other clostridial organisms Atsumiet al., Metab Eng 10:305-311 (2008); Boynton et al., J Bacteriol.178:3015-3024 (1996); Hillmer et al., FEBS Lett. 21:351-354 (1972)).Additional enoyl-CoA hydratase candidates are phaA and phaB, of P.putida, and paaA and paaB from P. fluorescens (Olivera et al., Proc.Natl. Acad. Sci U.S.A 95:6419-6424 (1998)). The gene product of pimF inRhodopseudomonas palustris is predicted to encode an enoyl-CoA hydratasethat participates in pimeloyl-CoA degradation (Harrison et al.,Microbiology 151:727-736 (2005)). Lastly, a number of Escherichia coligenes have been shown to demonstrate enoyl-CoA hydratase functionalityincluding maoC (Park et al., J Bacteriol. 185:5391-5397 (2003)), paaF(Ismail et al., Eur. J Biochem. 270:3047-3054 (2003); Park et al., Appl.Biochem. Biotechnol 113-116:335-346 (2004); Park et al., BiotechnolBioeng 86:681-686 (2004)) and paaG (Ismail et al., Eur. J Biochem.270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol113-116:335-346 (2004); Park and Yup, Biotechnol Bioeng 86:681-686(2004)). Enzymes with 3-hydroxyacyl-CoA dehydratase activity in S.cerevisiae include PHS1 and FOX2.

GenBank Gene Accession No. GI No. Organism ech NP_745498.1 26990073Pseudomonas putida crt NP_349318.1 15895969 Clostridium acetobutylicumcrt1 YP_001393856 153953091 Clostridium kluyveri phaA ABF82233.126990002 Pseudomonas putida phaB ABF82234.1 26990001 Pseudomonas putidapaaA NP_745427.1 106636093 Pseudomonas fluorescens paaB NP_745426.1106636094 Pseudomonas fluorescens pimF CAE29158.1 39650635Rhodopseudomonas palustris maoC NP_415905.1 16129348 Escherichia colipaaF NP_415911.1 16129354 Escherichia coli paaG NP_415912.1 16129355Escherichia coli FOX2 NP_012934.1 6322861 Saccharomyces cerevisiae PHS1NP_012438.1 6322364 Saccharomyces cerevisiae

Enoyl-CoA hydratases involved in beta-oxidation can also be used in anfatty alcohol, fatty aldehyde and fatty acid biosynthetic pathway. Forexample, the multifunctional MFP2 gene product of Arabidopsis thalianaexhibits an enoyl-CoA reductase activity selective for chain lengthsless than or equal to C14 (Arent et al, J Biol Chem 285:24066-77(2010)). Alternatively, the E. coli gene products of fadA and fadBencode a multienzyme complex involved in fatty acid oxidation thatexhibits enoyl-CoA hydratase activity (Yang et al., Biochemistry30:6788-6795 (1991); Yang, J Bacteriol. 173:7405-7406 (1991);Nakahigashi et al., Nucleic Acids Res. 18:4937 (1990)). The fadI andfadJ genes encode similar functions and are naturally expressed underanaerobic conditions (Campbell et al., Mol. Microbiol 47:793-805(2003)).

Protein GenBank ID GI Number Organism MFP2 AAD18042.1 4337027Arabidopsis thaliana fadA YP_026272.1 49176430 Escherichia coli fadBNP_418288.1 16131692 Escherichia coli fadI NP_416844.1 16130275Escherichia coli fadJ NP_416843.1 16130274 Escherichia coli fadRNP_415705.1 16129150 Escherichia coli

Chain length specificity of selected 3-hydroxyacyl-CoA dehydrataseenzymes is shown below.

Chain length Gene Organism C4-C6 crt Clostridium acetobutylicum C4-C7pimF Rhodopseudomonas palustris C4-C14 MFP2 Arabidopsis thaliana

Step D. Enoyl-CoA Reductase

Enoyl-CoA reductases (also known as acyl-CoA dehydrogenases,trans-2-enoyl-CoA reductases, or acyl-CoA oxidoreductases) catalyze theconversion of an enoyl-CoA to an acyl-CoA (step D of FIGS. 1 and 6).Exemplary acyl-CoA dehydrogenase or enoyl-CoA reductase (ECR) enzymesare the gene products of fadE of E. coli and Salmonella enterica (Iramet al, J Bacteriol 188:599-608 (2006)). The bcd gene product fromClostridium acetobutylicum (Atsumi et al., 10:305-311 (2008); Boynton etal., J Bacteriol. 178:3015-3024 (1996)) catalyzes the reduction ofcrotonyl-CoA to butyryl-CoA (EC 1.3.99.2). This enzyme participates inthe acetyl-CoA fermentation pathway to butyrate in Clostridial species(Jones et al., Microbiol Rev. 50:484-524 (1986)). Activity ofbutyryl-CoA reductase can be enhanced by expressing bcd in conjunctionwith expression of the C. acetobutylicum etfAB genes, which encode anelectron transfer flavoprotein. An additional candidate for theenoyl-CoA reductase step is the enoyl-CoA reductase (EC 1.3.1.44) TERfrom E. gracilis (Hoffmeister et al., J Biol. Chem 280:4329-4338(2005)). A construct derived from this sequence following the removal ofits mitochondrial targeting leader sequence was cloned in E. coliresulting in an active enzyme. A close homolog of the ECR protein fromthe prokaryote Treponema denticola, encoded by TDE0597, has also beencloned and expressed in E. coli (Tucci et al., FEBS Lett, 581:1561-1566(2007)). Six genes in Syntrophus aciditrophicus were identified bysequence homology to the C. acetobutylicum bcd gene product. The S.aciditrophicus genes syn_(—)02637 and syn_(—)02636 bear high sequencehomology to the etfAB genes of C. acetobutylicum, and are predicted toencode the alpha and beta subunits of an electron transfer flavoprotein.

Protein GenBank ID GI Number Organism fadE AAC73325.2 87081702Escherichia coli fadE YP_005241256.1 379699528 Salmonella enterica bcdNP_349317.1 15895968 Clostridium acetobutylicum etfA NP_349315.115895966 Clostridium acetobutylicum etfB NP_349316.1 15895967Clostridium acetobutylicum TER Q5EU90.1 62287512 Euglena gracilis TERNP_612558.1 19924091 Rattus norvegicus TDE0597 NP_971211.1 42526113Treponema denticola syn_02587 ABC76101 85721158 Syntrophusaciditrophicus syn_02586 ABC76100 85721157 Syntrophus aciditrophicussyn_01146 ABC76260 85721317 Syntrophus aciditrophicus syn_00480 ABC7789985722956 Syntrophus aciditrophicus syn_02128 ABC76949 85722006Syntrophus aciditrophicus syn_01699 ABC78863 85723920 Syntrophusaciditrophicus syn_02637 ABC78522.1 85723579 Syntrophus aciditrophicussyn_02636 ABC78523.1 85723580 Syntrophus aciditrophicus

Additional enoyl-CoA reductase enzyme candidates are found in organismsthat degrade aromatic compounds. Rhodopseudomonas palustris, a modelorganism for benzoate degradation, has the enzymatic capability todegrade pimelate via beta-oxidation of pimeloyl-CoA. Adjacent genes inthe pim operon, pimC and pimD, bear sequence homology to C.acetobutylicum bcd and are predicted to encode a flavin-containingpimeloyl-CoA dehydrogenase (Harrison et al., 151:727-736 (2005)). Thegenome of nitrogen-fixing soybean symbiont Bradyrhizobium japonicum alsocontains a pim operon composed of genes with high sequence similarity topimC and pimD of R. palustris (Harrison and Harwood, Microbiology151:727-736 (2005)).

Protein GenBank ID GI Number Organism pimC CAE29155 39650632Rhodopseudomonas palustris pimD CAE29154 39650631 Rhodopseudomonaspalustris pimC BAC53083 27356102 Bradyrhizobium japonicum pimD BAC5308227356101 Bradyrhizobium japonicum

An additional candidate is 2-methyl-branched chain enoyl-CoA reductase(EC 1.3.1.52 and EC 1.3.99.12), an enzyme catalyzing the reduction ofsterically hindered trans-enoyl-CoA substrates. This enzyme participatesin branched-chain fatty acid synthesis in the nematode Ascaris suum andis capable of reducing a variety of linear and branched chain substratesincluding 2-methylvaleryl-CoA, 2-methylbutanoyl-CoA,2-methylpentanoyl-CoA, octanoyl-CoA and pentanoyl-CoA (Duran et al.,268:22391-22396 (1993)). Two isoforms of the enzyme, encoded by genesacad1 and acad, have been characterized.

Protein GenBank ID GI Number Organism acad1 AAC48316.1 2407655 Ascarissuum acad AAA16096.1 347404 Ascaris suum

At least three mitochondrial enoyl-CoA reductase enzymes exist in E.gracilis and are applicable for use in the invention. Threemitochondrial enoyl-CoA reductase enzymes of E. gracilis (ECRL-3)exhibit different chain length preferences (Inui et al., EuropeanJournal of Biochemistry 142:121-126 (1984)), which is particularlyuseful for dictating the chain length of the desired fatty alcohol,fatty aldehyde or fatty acid products. EST's ELL00002199, ELL00002335,and ELL00002648, which are all annotated as mitochondrialtrans-2-enoyl-CoA reductases, can be used to isolate these additionalenoyl-CoA reductase genes by methods known in the art. Two ECR enzymesfrom rat liver microsomes also exhibit different substrate specificities(Nagi et al, Arch Biochem Biophys 226:50-64 (1983)). The sequences ofthese enzymes have not been identified to date. The Mycobacteriumsmegmatis enoyl-CoA reductase accepts acyl-CoA substrates of chainlengths between C10-C16 (Shimakata et al, J Biochem 89:1075-80 (1981)).

Enoyl-CoA reductases and their chain length specificities are shown inthe table below.

Chain length Gene Organism C4-C6 ECR1 Euglena gracilis C6-C8 ECR3Euglena gracilis C8-10 ECR2 Euglena gracilis C8-C16 Long chain ECRRattus norvegicus C10-C16 ECR Mycobacterium smegmatis C2-C18 fadESalmonella enterica

Step E. Acyl-CoA Reductase (Aldehyde Forming)

Reduction of an acyl-CoA to a fatty alcohol is catalyzed by either asingle enzyme or pair of enzymes that exhibit acyl-CoA reductase andalcohol dehydrogenase activities. Acyl-CoA dehydrogenases that reduce anacyl-CoA to its corresponding aldehyde include fatty acyl-CoA reductase(EC 1.2.1.42, 1.2.1.50), succinyl-CoA reductase (EC 1.2.1.76),acetyl-CoA reductase, butyryl-CoA reductase and propionyl-CoA reductase(EC 1.2.1.3). Aldehyde forming acyl-CoA reductase enzymes withdemonstrated activity on acyl-CoA, 3-hydroxyacyl-CoA and 3-oxoacyl-CoAsubstrates are known in the literature. Several acyl-CoA reductaseenzymes are active on 3-hydroxyacyl-CoA substrates. For example, somebutyryl-CoA reductases from Clostridial organisms, are active on3-hydroxybutyryl-CoA and propionyl-CoA reductase of L. reuteri is activeon 3-hydroxypropionyl-CoA. An enzyme for converting 3-oxoacyl-CoAsubstrates to their corresponding aldehydes is malonyl-CoA reductase.Enzymes in this class that demonstrate activity on enoyl-CoA substrateshave not been identified to date. Specificity for a particular substratecan be refined using evolution or enzyme engineering methods known inthe art.

Exemplary fatty acyl-CoA reductases enzymes are encoded by acr1 ofAcinetobacter calcoaceticus (Reiser, Journal of Bacteriology179:2969-2975 (1997)) and Acinetobacter sp. M-1 (Ishige et al., Appl.Environ. Microbiol. 68:1192-1195 (2002)). Two gene products fromMycobacterium tuberculosis accept longer chain fatty acyl-CoA substratesof length C16-C18 (Harminder Singh, U. Central Florida (2007)). Yetanother fatty acyl-CoA reductase is LuxC of Photobacterium phosphoreum(Lee et al, Biochim Biohys Acta 1388:215-22 (1997)). Enzymes withsuccinyl-CoA reductase activity are encoded by sucD of Clostridiumkluyveri (Sohling, J. Bacteriol. 178:871-880 (1996)) and sucD of P.gingivalis (Takahashi, J. Bacteriol 182:4704-4710 (2000)). Additionalsuccinyl-CoA reductase enzymes participate in the3-hydroxypropionate/4-hydroxybutyrate cycle of thermophilic archaeaincluding Metallosphaera sedula (Berg et al., Science 318:1782-1786(2007)) and Thermoproteus neutrophilus (Ramos-Vera et al., J Bacteriol,191:4286-4297 (2009)). The M. sedula enzyme, encoded by Msed_(—)0709, isstrictly NADPH-dependent and also has malonyl-CoA reductase activity.The T. neutrophilus enzyme is active with both NADPH and NADH. Theenzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encodedby bphG, is yet another as it has been demonstrated to oxidize andacylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehydeand formaldehyde (Powlowski, J. Bacteriol. 175:377-385 (1993)). Inaddition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhEin Leuconostoc mesenteroides has been shown to oxidize the branchedchain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya, J. Gen.Appl. Microbiol. 18:43-55 (1972); and Koo et al., Biotechnol Lett.27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similarreaction, conversion of butyryl-CoA to butyraldehyde, in solventogenicorganisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al.,Biosci Biotechnol Biochem., 71:58-68 (2007)). Exemplary propionyl-CoAreductase enzymes include pduP of Salmonella typhimurium LT2 (Leal,Arch. Microbiol. 180:353-361 (2003)) and eutE from E. coli (Skraly, WOPatent No. 2004/024876). The propionyl-CoA reductase of Salmonellatyphimurium LT2, which naturally converts propionyl-CoA topropionaldehyde, also catalyzes the reduction of 5-hydroxyvaleryl-CoA to5-hydroxypentanal (WO 2010/068953A2). The propionaldehyde dehydrogenaseof Lactobacillus reuteri, PduP, has a broad substrate range thatincludes butyraldehyde, valeraldehyde and 3-hydroxypropionaldehyde (Luoet al, Appl Microbiol Biotech, 89: 697-703 (2011). Additionally, someacyl-ACP reductase enzymes such as the orf1594 gene product ofSynechococcus elongatus PCC7942 also exhibit aldehyde-forming acyl-CoAreductase activity (Schirmer et al, Science, 329: 559-62 (2010)).Acyl-ACP reductase enzymes and homologs are described in further detailin Example IX.

Protein GenBank ID GI Number Organism acr1 YP_047869.1 50086359Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyiacr1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1 Rv1543 NP_216059.115608681 Mycobacterium tuberculosis Rv3391 NP_217908.1 15610527Mycobacterium tuberculosis LuxC AAT00788.1 46561111 Photobacteriumphosphoreum Msed_0709 YP_001190808.1 146303492 Metallosphaera sedulaTneu_0421 ACB39369.1 170934108 Thermoproteus neutrophilus sucD P38947.1172046062 Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonasgingivalis bphG BAA03892.1 425213 Pseudomonas sp adhE AAV66076.155818563 Leuconostoc mesenteroides bld AAP42563.1 31075383 Clostridiumsaccharoperbutylacetonicum pduP NP_460996 16765381 Salmonellatyphimurium LT2 eutE NP_416950 16130380 Escherichia coli pduP CCC03595.1337728491 Lactobacillus reuteri

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archaeal bacteria (Berg, Science 318:1782-1786 (2007);and Thauer, Science 318:1732-1733 (2007)). The enzyme utilizes NADPH asa cofactor and has been characterized in Metallosphaera and Sulfolobussp. (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Hugler, J.Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed_(—)0709in Metallosphaera sedula (Alber et al., J. Bacteriol. 188:8551-8559(2006); and Berg, Science 318:1782-1786 (2007)). A gene encoding amalonyl-CoA reductase from Sulfolobus tokodaii was cloned andheterologously expressed in E. coli (Alber et al., J. Bacteriol188:8551-8559 (2006). This enzyme has also been shown to catalyze theconversion of methylmalonyl-CoA to its corresponding aldehyde(WO2007141208 (2007)). Although the aldehyde dehydrogenase functionalityof these enzymes is similar to the bifunctional dehydrogenase fromChloroflexus aurantiacus, there is little sequence similarity. Bothmalonyl-CoA reductase enzyme candidates have high sequence similarity toaspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reductionand concurrent dephosphorylation of aspartyl-4-phosphate to aspartatesemialdehyde. Additional gene candidates can be found by sequencehomology to proteins in other organisms including Sulfolobussolfataricus and Sulfolobus acidocaldarius and have been listed below.Yet another candidate for CoA-acylating aldehyde dehydrogenase is theald gene from Clostridium beijerinckii (Toth, Appl. Environ. Microbiol.65:4973-4980 (1999). This enzyme has been reported to reduce acetyl-CoAand butyryl-CoA to their corresponding aldehydes. This gene is verysimilar to eutE that encodes acetaldehyde dehydrogenase of Salmonellatyphimurium and E. coli (Toth, Appl. Environ. Microbiol. 65:4973-4980(1999).

Gene GenBank ID GI Number Organism Msed_0709 YP_001190808.1 146303492Metallosphaera sedula mcr NP_378167.1 15922498 Sulfolobus tokodaii asd-2NP_343563.1 15898958 Sulfolobus solfataricus Saci_2370 YP_256941.170608071 Sulfolobus acidocaldarius Ald AAT66436 49473535 Clostridiumbeijerinckii eutE AAA80209 687645 Salmonella typhimurium eutE NP_41695016130380 Escherichia coli

Chain length specificity ranges of selected aldehyde-forming acyl-CoAreductase enzymes are show in the table below.

Chain length Gene Organism C2-C4 bphG Pseudomonas sp C4 Bld Clostridiumsaccharoperbutylacetonicum C12-C20 ACR Acinetobacter calcoaceticusC14-C18 Acr1 Acinetobacter sp. Strain M-1 C16-C18 Rv1543, Rv3391Mycobacterium tuberculosis

Step G. Acyl-CoA Reductase (Alcohol Forming)

Bifunctional alcohol-forming acyl-CoA reductase enzymes catalyze step G(i.e. step E and F) of FIGS. 1 and 6. Enzymes with this activity includeadhE of E. coli (Kessler et al., FEBS. Lett. 281:59-63 (1991))) andadhE2 of Clostridium acetobutylicum (Fontaine et al., J. Bacteriol.184:821-830 (2002))). The E. coli enzyme is active on C2 substrates,whereas the C. acetobutylicum enzyme has a broad substrate range thatspans C2-C8 (Dekishima et al, J Am Chem Soc 133:11399-11401 (2011)). TheC. acetobutylicum enzymes encoded by bdh I and bdh II (Walter, et al.,J. Bacteriol. 174:7149-7158 (1992)), reduce acetyl-CoA and butyryl-CoAto ethanol and butanol, respectively. The adhE gene produce fromLeuconostoc mesenteroides is active on acetyl-CoA and isobutyryl-CoA(Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al.,Biotechnol Lett, 27:505-510 (2005)). Enzyme candidates in otherorganisms including Roseiflexus castenholzii, Erythrobacter sp. NAP1 andmarine gamma proteobacterium HTCC2080 can be inferred by sequencesimilarity. Longer chain acyl-CoA molecules can be reduced to theircorresponding alcohols by enzymes such as the jojoba (Simmondsiachinensis) FAR which encodes an alcohol-forming fatty acyl-CoAreductase. Its overexpression in E. coli resulted in FAR activity andthe accumulation of C16-C18 fatty alcohols (Metz et al., Plant Physiol,122:635-644 (2000)). FAR enzymes in Arabidopsis thaliana include thegene products of At3g11980 and At3g44560 (Doan et al, J Plant Physiol166 (2006)). Bifunctional prokaryotic FAR enzymes are found inMarinobacter aquaeolei VT8 (Hofvander et al, FEBS Lett 3538-43 (2011)),Marinobacter algicola and Oceanobacter strain RED65 (US Pat Appl20110000125). Other suitable enzymes include bfar from Bombyx mori,mfar1 and mfar2 from Mus musculus; mfar2 from Mus musculus; acrM1 fromAcinetobacter sp. M1; and hfar from H. sapiens.

Protein GenBank ID GI Number Organism adhE NP_415757.1 16129202Escherichia coli adhE2 AAK09379.1 12958626 Clostridium acetobutylicumbdh I NP_349892.1 15896543 Clostridium acetobutylicum bdh II NP_349891.115896542 Clostridium acetobutylicum adhE AAV66076.1 55818563 Leuconostocmesenteroides mcr AAS20429.1 42561982 Chloroflexus aurantiacus Rcas_2929YP_001433009.1 156742880 Roseiflexus castenholzii NAP1_02720ZP_01039179.1 85708113 Erythrobacter sp. NAP1 MGP2080_00535ZP_01626393.1 119504313 marine gamma proteobacterium HTCC2080 FARAAD38039.1 5020215 Simmondsia chinensis At3g11980 NP_191229.1 15228993Arabidopsis thaliana At3g44560 NP_190042.2 145339120 Arabidopsisthaliana FAR YP_959486.1 120555135 Marinobacter aquaeolei bfar Q8R07981901336 Bombyx mori

Chain length specificity ranges of selected alcohol-forming acyl-CoAreductase enzymes are show in the table below.

Chain length Gene Organism C2 adhE Escherichia coli C2-C8 adhe2Clostridium acetobutylicum C14-C16 At3g11980 Arabidopsis thaliana C16At3g44560 Arabidopsis thaliana C16-C18 FAR Simmondsia chinensis C14-C18FAR Marinobacter aquaeolei

Step F. Fatty Aldehyde Reductase

Exemplary genes encoding enzymes that catalyze the conversion of analdehyde to alcohol (i.e., alcohol dehydrogenase or equivalentlyaldehyde reductase) include alrA encoding a medium-chain alcoholdehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol.66:5231-5235 (2000)), yqhD and fucO from E. coli (Sulzenbacher et al.,342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicum whichconverts butyryaldehyde into butanol (Walter et al., J Bacteriol174:7149-7158 (1992)). The alrA gene product showed no activity onaldehydes longer than C14, and favored the reductive direction (Tani etal, supra). YqhD catalyzes the reduction of a wide range of aldehydesusing NADPH as the cofactor, with a preference for chain lengths longerthan C(3) (Sulzenbacher et al, J Mol Biol 342:489-502 (2004); Perez etal., J Biol. Chem. 283:7346-7353 (2008)). The adhA gene product fromZymomonas mobilis has been demonstrated to have activity on a number ofaldehydes including formaldehyde, acetaldehyde, propionaldehyde,butyraldehyde, and acrolein (Kinoshita et al., Appl Microbiol Biotechnol22:249-254 (1985)). Additional aldehyde reductase candidates are encodedby bdh in C. saccharoperbutylacetonicum and Cbei_(—)1722, Cbei_(—)2181and Cbei_(—)2421 in C. beijerinckii. The alcohol dehydrogenase fromLeifsonia sp. S749 shows maximal activity on medium chain-lengthsubstrates of length C6-C7 (Inoue et al, AEM 71: 3633-3641 (2005). Theadh gene product of Pseudomonas putida is active on substrates of lengthC3-C10 (Nagashima et al, J Ferment Bioeng 82:328-33(1996)). The alcoholdehydrogenase enzymes ADH1 and ADH2 of Geobacillus thermodenitrificansoxidize alcohols up to a chain length of C30 (Liu et al, Physiol Biochem155:2078-85 (2009)).

Protein GenBank ID GI Number Organism alrA BAB12273.1 9967138Acinetobacter sp. strain M-1 ADH2 NP_014032.1 6323961 Saccharomycescerevisiae yqhD NP_417484.1 16130909 Escherichia coli fucO NP_417279.116130706 Escherichia coli bdh I NP_349892.1 15896543 Clostridiumacetobutylicum bdh II NP_349891.1 15896542 Clostridium acetobutylicumadhA YP_162971.1 56552132 Zymomonas mobilis bdh BAF45463.1 124221917Clostridium saccharoperbutylacetonicum Cbei_1722 YP_001308850 150016596Clostridium beijerinckii Cbei_2181 YP_001309304 150017050 Clostridiumbeijerinckii Cbei_2421 YP_001309535 150017281 Clostridium beijerinckiilsadh BAD99642.1 67625613 Leifsonia sp. S749 adh Pseudomonas putida

Native alcohol dehydrogenases also convert aldehyde substrates toalcohol products. To date, seven alcohol dehydrogenases, ADHI-ADHVII,have been reported in S. cerevisiae (de Smidt et al, FEMS Yeast Res8:967-78 (2008)). ADH1 (GI:1419926) is the key enzyme responsible forreducing acetaldehyde to ethanol in the cytosol under anaerobicconditions. In K. lactis, two NAD-dependent cytosolic alcoholdehydrogenases have been identified and characterized. These genes alsoshow activity for other aliphatic alcohols. The genes ADH1 (GI:113358)and ADHII (GI:51704293) are preferentially expressed in glucose-growncells (Bozzi et al, Biochim Biophys Acta 1339:133-142 (1997)). Cytosolicalcohol dehydrogenases are encoded by ADH1 (GI:608690) in C. albicans,ADH1 (GI:3810864) in S. pombe, ADH1 (GI:5802617) in Y. lipolytica, ADH1(GI:2114038) and ADHII (GI:2143328) in Pichia stipitis orScheffersomyces stipitis (Passoth et al, Yeast 14:1311-23 (1998)).Candidate alcohol dehydrogenases are shown the table below.

Protein GenBank ID GI number Organism SADH BAA24528.1 2815409 Candidaparapsilosis ADH1 NP_014555.1 6324486 Saccharomyces cerevisiae s288cADH2 NP_014032.1 6323961 Saccharomyces cerevisiae s288c ADH3 NP_013800.16323729 Saccharomyces cerevisiae s288c ADH4 NP_011258.2 269970305Saccharomyces cerevisiae s288c ADH5 (SFA1) NP_010113.1 6320033Saccharomyces cerevisiae s288c ADH6 NP_014051.1 6323980 Saccharomycescerevisiae s288c ADH7 NP_010030.1 6319949 Saccharomyces cerevisiae s288cadhP CAA44614.1 2810 Kluyveromyces lactis ADH1 P20369.1 113358Kluyveromyces lactis ADH2 CAA45739.1 2833 Kluyveromyces lactis ADH3P49384.2 51704294 Kluyveromyces lactis ADH1 YP_001126968.1 138896515Geobacillus thermodenitrificans ADH2 YP_001125863.1 138895410Geobacillus thermodenitrificans

Substrate specificity ranges of selected alcohol dehydrogenase enzymesare show in the table below.

Chain length Gene Organism C6-C7 lsadh Leifsonia sp. S749 C2-C8 yqhDEscherichia coli C3-C10 Adh Pseudomonas putida C2-C14 alrA Acinetobactersp. strain M-1 C2-C30 ADH1 Geobacillus thermodenitrificans

Step O. Elongase

Elongase (ELO) enzymes utilize malonyl-CoA to add a C2 unit to a growingacyl-CoA chain. This process also involves decarboxylation and is thuslargely irreversible. Trypanosoma brucei, a eukaryotic human parasite,is known to produce long chain fatty acids using an elongase system. Theprocess is initiated by butyryl-CoA. In particular, the ELO systemesterifies the growing fatty acid chain to CoA intermediates rather thanACP intermediates like the bacterial and other microbial counterparts(Lee et al, Cell 126, 691-699, 2006; Cronan, Cell, 126, 2006). This isin contrast to typical bacterial fatty acid elongation which isinitiated following the formation of acetoacetyl acyl-ACP frommalonyl-ACP. So far, four ELOs (encoded by ELO1-4) that are homologousto their animal counterparts have been found in T brucei (Lee et al,Nature Reviews Microbiology, Vol 5, 287-297, 2007). ELO1-3 togetheraccount for synthesis of saturated fatty acids up to a chain length ofC18. ELO1 converts C4 to C10, ELO2 extends the chain length from C10 tomyristate (C14), and ELO3 extends myristate to C18. There is someoverlap in ELO specificity; for example, ELO1 can extend a C10 primer toC12, albeit with low activity. ELO4 is an example of an ELO that isspecific for poly unsaturated fatty acids (PUFAs). It extendsarachidonate (C20:4) by two carbon atoms. Several additional ELO enzymescan be found by sequence homology (see Lee et al, Nature ReviewsMicrobiology, Vol 5, 287-297, 2007).

Elongase enzymes are found in several compartments including themitochondria, endoplasmic reticulum, proteoliposomes and peroxisomes.For example, some yeast such as S. cerevisiae are able to synthesizelong-chain fatty acids of chain length C16 and higher via amitochondrial elongase which accepts exogenous or endogenous acyl-CoAsubstrates (Bessoule et al, FEBS Lett 214: 158-162 (1987)). This systemrequires ATP for activity. The endoplasmic reticulum also has anelongase system for synthesizing very long chain fatty acids (C18+) fromacyl-CoA substrates of varying lengths (Kohlwein et al, Mol Cell Biol21:109-25 (2001)). Genes involved in this system include TSC13, ELO2 andELO3. ELO1 catalyzes the elongation of C12 acyl-CoAs to C16-C18 fattyacids.

Protein Accession # GI number Organism ELO2 NP_009963.1 6319882Saccharomyces cerevisiae ELO3 NP_013476.3 398366027 Saccharomycescerevisiae TSC13 NP_010269.1 6320189 Saccharomyces cerevisiae ELO1NP_012339.1 6322265 Saccharomyces cerevisiae ELO1 AAX70671.1 62176566Trypanosoma brucei ELO2 AAX70672.1 62176567 Trypanosoma brucei ELO3AAX70673.1 62176568 Trypanosoma brucei ELO4 AAX70768.1 62176665Trypanosoma brucei ELO4 AAX69821.1 62175690 Trypanosoma brucei

Those skilled in the art also can obtain nucleic acids encoding any orall of the malonyl-CoA independent FAS pathway or acyl-reduction pathwayenzymes by cloning using known sequences from available sources. Forexample, any or all of the encoding nucleic acids for the malonyl-CoAindependent FAS pathway can be readily obtained using methods well knownin the art from E. gracilis as this pathway has been well characterizedin this organism. E. gracilis encoding nucleic acids can be isolatedfrom, for example, an E. gracilis cDNA library using probes of knownsequence. The probes can be designed with whole or partial DNA sequencesfrom the following EST sequences from the publically available sequencedatabase TBestDB (http://tbestdb.bcm.umontreal.ca). The nucleic acidsgenerated from this process can be inserted into an appropriateexpression vector and transformed into E. coli or other microorganismsto generate fatty alcohols, fatty aldehydes or fatty acids productionorganisms of the invention.

Thiolase (FIG. 1A): ELL00002550, ELL00002493, ELL00000789

3-Hydroxyacyl-CoA dehydrogenase (FIG. 1B): ELL00000206, ELL00002419,ELL00006286, ELL00006656

Enoyl-CoA hydratase (FIG. 1C): ELL00005926, ELL00001952, ELL00002235,ELL00006206

Enoyl-CoA reductase (FIG. 1D): ELL00002199, ELL00002335, ELL00002648

Acyl-CoA reductase (FIG. 1E; 1E/F): ELL00002572, ELL00002581,ELL00000108

Alternatively, the above EST sequences can be used to identify homologuepolypeptides in GenBank through BLAST search. The resulting homologuepolypeptides and their corresponding gene sequences provide additionalencoding nucleic acids for transformation into E. coli or othermicroorganisms to generate the fatty alcohols, fatty aldehydes or fattyacids producing organisms of the invention. Listed below are exemplaryhomologue polypeptide and their gene accession numbers in GenBank whichare applicable for use in the non-naturally occurring organisms of theinvention. Ketoacyl-CoA acyltransferase (or ketoacyl-CoA thiolase)

Protein GenBank ID GI number Organism Dole_2160 YP_001530041 158522171Desulfococcus oleovorans Hxd3 DalkDRAFT_1939 ZP_02133627 163726110Desulfatibacillum alkenivorans AK-01 BSG1_09488 ZP_01860900 149182424Bacillus sp. SG-1

3-Hydroxyacyl-CoA Dehydrogenase

Protein GenBank ID GI number Organism AaeL_AAEL002841 XP_001655993157132312 Aedes aegypti hadh NP_001011073 58331907 Xenopus tropicalishadh NP_001003515 51011113 Danio rerio

Enoyl-CoA hydratase

Protein GenBank ID GI number Organism Tb927.3.4850 XP_844077 72387305Trypanosoma brucei Tc00.1047053509701.10 XP_802711 71399112 Trypanosomacruzi strain CL Brener PputGB1_3629 YP_001669856 167034625 Pseudomonasputida GB-1

Enoyl-CoA reductase

Protein GenBank ID GI number Organism mecr XP_642118 66816217Dictyostelium discoideum AX4 NEMVEDRAFT_v1g228294 XP_001639469 156402181Nematostella vectensis AaeL_AAEL003995 XP_001648220 157104018 Aedesaegypti

In addition to the above exemplary encoding nucleic acids, nucleic acidsother than those within the MI-FAE cycle, MD-FAE and/or terminationpathways of the invention also can be introduced into a host organismfor further production of fatty alcohols, fatty aldehydes or fattyacids. For example, the Ralstonia eutropha BktB and PhbB genes catalyzethe condensation of butyryl-CoA and acetyl-CoA to formβ-keto-hexanoyl-CoA and the reduction of β-keto-hexanoyl-CoA to3-hydroxy-hexanoyl-CoA (Fukui et al., Biomacromolecules 3:618-624(2002)). To improve the production of fatty alcohols, exogenous DNAsequences encoding for these specific enzymes can be expressed in theproduction host of interest. Furthermore, the above described enzymescan be subjected to directed evolution to generate improved versions ofthese enzymes with high activity and high substrate specificity. Asimilar approach also can be utilized with any or all other enzymaticsteps in the fatty alcohol, fatty aldehyde or fatty acid producingpathways of the invention to, for example, improve enzymatic activityand/or specificity and/or to generate a fatty alcohol, a fatty aldehydeor a fatty acid of a predetermined chain length or lengths.

Example II Pathways for Producing Cytosolic Acetyl-CoA from CytosolicPyruvate

The following example describes exemplary pathways for the conversion ofcytosolic pyruvate and threonine to cytosolic acetyl-CoA, as shown inFIG. 2.

Pathways for the conversion of cytosolic pyruvate and threonine tocytosolic acetyl-CoA could enable deployment of a cytosolic fattyalcohol, fatty aldehyde or fatty acid production pathway that originatesfrom acetyl-CoA. Several pathways for converting cytosolic pyruvate tocytosolic acetyl-CoA are shown in FIG. 2. Direct conversion of pyruvateto acetyl-CoA can be catalyzed by pyruvate dehydrogenase, pyruvateformate lyase, pyruvate:NAD(P) oxidoreductase or pyruvate:ferredoxinoxidoreductase. If a pyruvate formate lyase is utilized, the formatebyproduct can be further converted to CO2 by formate dehydrogenase orformate hydrogen lyase.

Indirect conversion of pyruvate to acetyl-CoA can proceed throughseveral alternate routes. Pyruvate can be converted to acetaldehyde by apyruvate decarboxylase. Acetaldehyde can then converted to acetyl-CoA byan acylating (CoA-dependent) acetaldehyde dehydrogenase. Alternately,acetaldehyde generated by pyruvate decarboxylase can be converted toacetyl-CoA by the “PDH bypass” pathway. In this pathway, acetaldehyde isoxidized by acetaldehyde dehydrogenase to acetate, which is thenconverted to acetyl-CoA by a CoA ligase, synthetase or transferase. Inanother embodiment, the acetate intermediate is converted by an acetatekinase to acetyl-phosphate that is then converted to acetyl-CoA by aphosphotransacetylase. In yet another embodiment, pyruvate is directlyconverted to acetyl-phosphate by a pyruvate oxidase (acetyl-phosphateforming). Conversion of pyruvate to acetate is also catalyzed byacetate-forming pyruvate oxidase.

Cytosolic acetyl-CoA can also be synthesized from threonine byexpressing a native or heterologous threonine aldolase (FIG. 5J) (vanMaris et al, AEM 69:2094-9 (2003)). Threonine aldolase convertsthreonine into acetaldehyde and glycine. The acetaldehyde product issubsequently converted to acetyl-CoA by various pathways describedabove.

Gene candidates for the acetyl-CoA forming enzymes shown in FIG. 2 aredescribed below.

Pyruvate oxidase (acetate-forming) (FIG. 2A) or pyruvate:quinoneoxidoreductase (PQO) can catalyze the oxidative decarboxylation ofpyruvate into acetate, using ubiquione (EC 1.2.5.1) or quinone (EC1.2.2.1) as an electron acceptor. The E. coli enzyme, PoxB, is localizedon the inner membrane (Abdel-Hamid et al., Microbiol 147:1483-98(2001)). The enzyme has thiamin pyrophosphate and flavin adeninedinucleotide (FAD) cofactors (Koland and Gennis, Biochemistry21:4438-4442 (1982)); O'Brien et al., Biochemistry 16:3105-3109 (1977);O'Brien and Gennis, J. Biol. Chem. 255:3302-3307 (1980)). PoxB hassimilarity to pyruvate decarboxylase of S. cerevisiae and Zymomonasmobilis. The pqo transcript of Corynebacterium glutamicum encodes aquinone-dependent and acetate-forming pyruvate oxidoreductase (Schreineret al., J Bacteriol 188:1341-50 (2006)). Similar enzymes can be inferredby sequence homology.

Protein GenBank ID GI Number Organism poxB NP_415392.1 16128839Escherichia coli pqo YP_226851.1 62391449 Corynebacterium glutamicumpoxB YP_309835.1 74311416 Shigella sonnei poxB ZP_03065403.1 194433121Shigella dysenteriae

The acylation of acetate to acetyl-CoA (FIG. 2B) can be catalyzed byenzymes with acetyl-CoA synthetase, ligase or transferase activity. Twoenzymes that can catalyze this reaction are AMP-forming acetyl-CoAsynthetase or ligase (EC 6.2.1.1) and ADP-forming acetyl-CoA synthetase(EC 6.2.1.13). AMP-forming acetyl-CoA synthetase (ACS) is thepredominant enzyme for activation of acetate to acetyl-CoA. ExemplaryACS enzymes are found in E. coli (Brown et al., J. Gen. Microbiol.102:327-336 (1977)), Ralstonia eutropha (Priefert and Steinbuchel, J.Bacteriol. 174:6590-6599 (1992)), Methanothermobacter thermautotrophicus(Ingram-Smith and Smith, Archaea 2:95-107 (2007)), Salmonella enterica(Gulick et al., Biochemistry 42:2866-2873 (2003)) and Saccharomycescerevisiae (Jogl and Tong, Biochemistry 43:1425-1431 (2004)).ADP-forming acetyl-CoA synthetases are reversible enzymes with agenerally broad substrate range (Musfeldt and Schonheit, J. Bacteriol.184:636-644 (2002)). Two isozymes of ADP-forming acetyl-CoA synthetasesare encoded in the Archaeoglobus fulgidus genome by are encoded byAF1211 and AF1983 (Musfeldt and Schonheit, supra (2002)). The enzymefrom Haloarcula marismortui (annotated as a succinyl-CoA synthetase)also accepts acetate as a substrate and reversibility of the enzyme wasdemonstrated (Brasen and Schonheit, Arch. Microbiol. 182:277-287(2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeonPyrobaculum aerophilum showed the broadest substrate range of allcharacterized ACDs, reacting with acetate, isobutyryl-CoA (preferredsubstrate) and phenylacetyl-CoA (Brasen and Schonheit, supra (2004)).Directed evolution or engineering can be used to modify this enzyme tooperate at the physiological temperature of the host organism. Theenzymes from A. fulgidus, H. marismortui and P. aerophilum have all beencloned, functionally expressed, and characterized in E. coli (Brasen andSchonheit, supra (2004); Musfeldt and Schonheit, supra (2002)).Additional candidates include the succinyl-CoA synthetase encoded bysucCD in E. coli (Buck et al., Biochemistry 24:6245-6252 (1985)) and theacyl-CoA ligase from Pseudomonas putida (Fernandez-Valverde et al.,Appl. Environ. Microbiol. 59:1149-1154 (1993)). The aforementionedproteins are shown below.

Protein GenBank ID GI Number Organism acs AAC77039.1 1790505 Escherichiacoli acoE AAA21945.1 141890 Ralstonia eutropha acs1 ABC87079.1 86169671Methanothermobacter thermautotrophicus acs1 AAL23099.1 16422835Salmonella enterica ACS1 Q01574.2 257050994 Saccharomyces cerevisiaeAF1211 NP_070039.1 11498810 Archaeoglobus fulgidus AF1983 NP_070807.111499565 Archaeoglobus fulgidus scs YP_135572.1 55377722 Haloarculamarismortui PAE3250 NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949Escherichia coli paaF AAC24333.2 22711873 Pseudomonas putida

The acylation of acetate to acetyl-CoA can also be catalyzed by CoAtransferase enzymes (FIG. 2B). Numerous enzymes employ acetate as theCoA acceptor, resulting in the formation of acetyl-CoA. An exemplary CoAtransferase is acetoacetyl-CoA transferase, encoded by the E. coli atoA(alpha subunit) and atoD (beta subunit) genes (Korolev et al., ActaCrystallogr. D. Biol. Crystallogr. 58:2116-2121 (2002); Vanderwinkel etal., 33:902-908 (1968)). This enzyme has a broad substrate range (Srameket al., Arch Biochem Biophys 171:14-26 (1975)) and has been shown totransfer the CoA moiety to acetate from a variety of branched and linearacyl-CoA substrates, including isobutyrate (Matthies et al., ApplEnviron. Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al.,Biochem. Biophys. Res. Commun. 33:902-908 (1968)) and butanoate(Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)).Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncanet al., 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al.,Appl Environ Microbiol 56:1576-1583 (1990); Wiesenborn et al., ApplEnviron Microbiol 55:323-329 (1989)), and Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem.71:58-68 (2007)).

Gene GI # Accession No. Organism atoA 2492994 P76459.1 Escherichia coliatoD 2492990 P76458.1 Escherichia coli actA 62391407 YP_226809.1Corynebacterium glutamicum cg0592 62389399 YP_224801.1 Corynebacteriumglutamicum ctfA 15004866 NP_149326.1 Clostridium acetobutylicum ctfB15004867 NP_149327.1 Clostridium acetobutylicum ctfA 31075384 AAP42564.1Clostridium saccharoperbutylacetonicum ctfB 31075385 AAP42565.1Clostridium saccharoperbutylacetonicum

Acetate kinase (EC 2.7.2.1) can catalyzes the reversible ATP-dependentphosphorylation of acetate to acetylphosphate (FIG. 2C). Exemplaryacetate kinase enzymes have been characterized in many organismsincluding E. coli, Clostridium acetobutylicum and Methanosarcinathermophila (Ingram-Smith et al., J. Bacteriol. 187:2386-2394 (2005);Fox and Roseman, J. Biol. Chem. 261:13487-13497 (1986); Winzer et al.,Microbioloy 143 (Pt 10):3279-3286 (1997)). Acetate kinase activity hasalso been demonstrated in the gene product of E. coli purT (Marolewskiet al., Biochemistry 33:2531-2537 (1994). Some butyrate kinase enzymes(EC 2.7.2.7), for example buk1 and buk2 from Clostridium acetobutylicum,also accept acetate as a substrate (Hartmanis, M. G., J. Biol. Chem.262:617-621 (1987)). Homologs exist in several other organisms includingSalmonella enterica and Chlamydomonas reinhardtii.

Protein GenBank ID GI Number Organism ackA NP_416799.1 16130231Escherichia coli Ack AAB18301.1 1491790 Clostridium acetobutylicum AckAAA72042.1 349834 Methanosarcina thermophila purT AAC74919.1 1788155Escherichia coli buk1 NP_349675 15896326 Clostridium acetobutylicum buk2Q97II1 20137415 Clostridium acetobutylicum ackA NP_461279.1 16765664Salmonella typhimurium ACK1 XP_001694505.1 159472745 Chlamydomonasreinhardtii ACK2 XP_001691682.1 159466992 Chlamydomonas reinhardtii

The formation of acetyl-CoA from acetyl-phosphate can be catalyzed byphosphotransacetylase (EC 2.3.1.8) (FIG. 2D). The pta gene from E. coliencodes an enzyme that reversibly converts acetyl-CoA intoacetyl-phosphate (Suzuki, T., Biochim. Biophys. Acta 191:559-569 (969)).Additional acetyltransferase enzymes have been characterized in Bacillussubtilis (Rado and Hoch, Biochim. Biophys. Acta 321:114-125 (1973),Clostridium kluyveri (Stadtman, E., Methods Enzymol. 1:5896-599 (1955),and Thermotoga maritima (Bock et al., J. Bacteriol. 181:1861-1867(1999)). This reaction can also be catalyzed by somephosphotranbutyrylase enzymes (EC 2.3.1.19), including the ptb geneproducts from Clostridium acetobutylicum (Wiesenborn et al., App.Environ. Microbiol. 55:317-322 (1989); Walter et al., Gene 134:107-111(1993)). Additional ptb genes are found in butyrate-producing bacteriumL2-50 (Louis et al., J. Bacteriol. 186:2099-2106 (2004) and Bacillusmegaterium (Vazquez et al., Curr. Microbiol. 42:345-349 (2001). Homologsto the E. coli pta gene exist in several other organisms includingSalmonella enterica and Chlamydomonas reinhardtii.

Protein GenBank ID GI Number Organism Pta NP_416800.1 71152910Escherichia coli Pta P39646 730415 Bacillus subtilis Pta A5N801146346896 Clostridium kluyveri Pta Q9X0L4 6685776 Thermotoga maritimePtb NP_349676 34540484 Clostridium acetobutylicum Ptb AAR19757.138425288 butyrate-producing bacterium L2-50 Ptb CAC07932.1 10046659Bacillus megaterium Pta NP_461280.1 16765665 Salmonella enterica subsp.enterica serovar Typhimurium str. LT2 PAT2 XP_001694504.1 159472743Chlamydomonas reinhardtii PAT1 XP_001691787.1 159467202 Chlamydomonasreinhardtii

Pyruvate decarboxylase (PDC) is a key enzyme in alcoholic fermentation,catalyzing the decarboxylation of pyruvate to acetaldehyde (FIG. 2E).The PDC1 enzyme from Saccharomyces cerevisiae has been extensivelystudied (Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001);Li et al., Biochemistry. 38:10004-10012 (1999); ter Schure et al., Appl.Environ. Microbiol. 64:1303-1307 (1998)). Other well-characterized PDCenzymes are found in Zymomonas mobilus (Siegert et al., Protein Eng DesSel 18:345-357 (2005)), Acetobacter pasteurians (Chandra et al.,176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al.,269:3256-3263 (2002)). The PDC1 and PDC5 enzymes of Saccharomycescerevisiae are subject to positive transcriptional regulation by PDC2(Hohmann et al, Mol Gen Genet 241:657-66 (1993)). Pyruvate decarboxylaseactivity is also possessed by a protein encoded by CTRG_(—)03826(GI:255729208) in Candida tropicalis, PDC1 (GI number: 1226007) inKluyveromyces lactis, YALI0D10131g (GI:50550349) in Yarrowia lipolytica,PAS_chr3_(—)0188 (GI:254570575) in Pichia pastoris, pyruvatedecarboxylase (GI: GI:159883897) in Schizosaccharomyces pombe,ANI_(—)1_(—)1024084 (GI:145241548), ANI_(—)1_(—)796114 (GI:317034487),ANI_(—)1_(—)936024 (GI:317026934) and ANI_(—)1_(—)2276014 (GI:317025935)in Aspergillus niger.

Protein GenBank ID GI Number Organism pdc P06672.1 118391 Zymomonasmobilis pdc1 P06169 30923172 Saccharomyces cerevisiae Pdc2 NP_010366.16320286 Saccharomyces cerevisiae Pdc5 NP_013235.1 6323163 Saccharomycescerevisiae CTRG_03826 XP_002549529 255729208 Candida tropicalis,CU329670.1: CAA90807 159883897 Schizosaccharomyces 585597 . . . 587312pombe YALI0D10131g XP_502647 50550349 Yarrowia lipolytica PAS_chr3_0188XP_002492397 254570575 Pichia pastoris pdc Q8L388 20385191 Acetobacterpasteurians pdc1 Q12629 52788279 Kluyveromyces lactis ANI_1_1024084XP_001393420 145241548 Aspergillus niger ANI_1_796114 XP_001399817317026934 Aspergillus niger ANI_1_936024 XP_001396467 317034487Aspergillus niger ANI_1_2276014 XP_001388598 317025935 Aspergillus niger

Aldehyde dehydrogenase enzymes in EC class 1.2.1 catalyze the oxidationof acetaldehyde to acetate (FIG. 2F). Exemplary genes encoding thisactivity were described above. The oxidation of acetaldehyde to acetatecan also be catalyzed by an aldehyde oxidase with acetaldehyde oxidaseactivity. Such enzymes can convert acetaldehyde, water and O₂ to acetateand hydrogen peroxide. Exemplary aldehyde oxidase enzymes that have beenshown to catalyze this transformation can be found in Bos taurus and Musmusculus (Garattini et al., Cell Mol Life Sci 65:1019-48 (2008); Cabreet al., Biochem Soc Trans 15:882-3 (1987)). Additional aldehyde oxidasegene candidates include the two flavin- and molybdenum-containingaldehyde oxidases of Zea mays, encoded by zmAO-1 and zmAO-2 (Sekimoto etal., J Biol Chem 272:15280-85 (1997)).

GenBank Gene Accession No. GI No. Organism zmAO-1 NP_001105308.1162458742 Zea mays zmAO-2 BAA23227.1 2589164 Zea mays Aox1 O54754.220978408 Mus musculus XDH DAA24801.1 296482686 Bos taurus

Pyruvate oxidase (acetyl-phosphate forming) can catalyze the conversionof pyruvate, oxygen and phosphate to acetyl-phosphate and hydrogenperoxide (FIG. 2G). This type of pyruvate oxidase is soluble andrequires the cofactors thiamin diphosphate and flavin adeninedinucleotide (FAD). Acetyl-phosphate forming pyruvate oxidase enzymescan be found in lactic acid bacteria Lactobacillus delbrueckii andLactobacillus plantarum (Lorquet et al., J Bacteriol 186:3749-3759(2004); Hager et al., Fed Proc 13:734-38 (1954)). A crystal structure ofthe L. plantarum enzyme has been solved (Muller et al., (1994)). InStreptococcus sanguinis and Streptococcus pneumonia, acetyl-phosphateforming pyruvate oxidase enzymes are encoded by the spxB gene(Spellerberg et al., Mol Micro 19:803-13 (1996); Ramos-Montanez et al.,Mol Micro 67:729-46 (2008)). The SpxR was shown to positively regulatethe transcription of spxB in S. pneumoniae (Ramos-Montanez et al.,supra). A similar regulator in S. sanguinis was identified by sequencehomology. Introduction or modification of catalase activity can reduceaccumulation of the hydrogen peroxide product.

GenBank Gene Accession No. GI No. Organism poxB NP_786788.1 28379896Lactobacillus plantarum spxB L39074.1 1161269 Streptococcus pneumoniaeSpd_0969 YP_816445.1 116517139 Streptococcus (spxR) pneumoniae spxBZP_07887723.1 315612812 Streptococcus sanguinis spxR ZP_07887944.1 GI:315613033 Streptococcus sanguinis

The pyruvate dehydrogenase (PDH) complex catalyzes the conversion ofpyruvate to acetyl-CoA (FIG. 2H). The E. coli PDH complex is encoded bythe genes aceEF and lpdA. Enzyme engineering efforts have improved theE. coli PDH enzyme activity under anaerobic conditions (Kim et al., J.Bacteriol. 190:3851-3858 (2008); Kim et al., Appl. Environ. Microbiol.73:1766-1771 (2007); Zhou et al., Biotechnol. Lett. 30:335-342 (2008)).In contrast to the E. coli PDH, the B. subtilis complex is active andrequired for growth under anaerobic conditions (Nakano et al.,179:6749-6755 (1997)). The Klebsiella pneumoniae PDH, characterizedduring growth on glycerol, is also active under anaerobic conditions(Menzel et al., 56:135-142 (1997)). Crystal structures of the enzymecomplex from bovine kidney (Zhou et al., 98:14802-14807 (2001)) and theE2 catalytic domain from Azotobacter vinelandii are available (Matteviet al., Science. 255:1544-1550 (1992)). Some mammalian PDH enzymescomplexes can react on alternate substrates such as 2-oxobutanoate.Comparative kinetics of Rattus norvegicus PDH and BCKAD indicate thatBCKAD has higher activity on 2-oxobutanoate as a substrate (Paxton etal., Biochem. J. 234:295-303 (1986)). The S. cerevisiae PDH complexcanconsist of an E2 (LAT1) core that binds E1 (PDA1, PDB1), E3 (LPD1),and Protein X (POX1) components (Pronk et al., Yeast 12:1607-1633(1996)). The PDH complex of S. cerevisiae is regulated byphosphorylation of E1 involving PKP1 (PDH kinase I), PTC5 (PDHphosphatase I), PKP2 and PTC6. Modification of these regulators may alsoenhance PDH activity. Coexpression of lipoyl ligase (LplA of E. coli andAIM22 in S. cerevisiae) with PDH in the cytosol may be necessary foractivating the PDH enzyme complex. Increasing the supply of cytosoliclipoate, either by modifying a metabolic pathway or mediasupplementation with lipoate, may also improve PDH activity.

Gene Accession No. GI Number Organism aceE NP_414656.1 16128107Escherichia coli aceF NP_414657.1 16128108 Escherichia coli lpdNP_414658.1 16128109 Escherichia coli lplA NP_418803.1 16132203Escherichia coli pdhA P21881.1 3123238 Bacillus subtilis pdhB P21882.1129068 Bacillus subtilis pdhC P21883.2 129054 Bacillus subtilis pdhDP21880.1 118672 Bacillus subtilis aceE YP_001333808.1 152968699Klebsiella pneumoniae aceF YP_001333809.1 152968700 Klebsiellapneumoniae lpdA YP_001333810.1 152968701 Klebsiella pneumoniae Pdha1NP_001004072.2 124430510 Rattus norvegicus Pdha2 NP_446446.1 16758900Rattus norvegicus Dlat NP_112287.1 78365255 Rattus norvegicus DldNP_955417.1 40786469 Rattus norvegicus LAT1 NP_014328 6324258Saccharomyces cerevisiae PDA1 NP_011105 37362644 Saccharomycescerevisiae PDB1 NP_009780 6319698 Saccharomyces cerevisiae LPD1NP_116635 14318501 Saccharomyces cerevisiae PDX1 NP_011709 6321632Saccharomyces cerevisiae AIM22 NP_012489.2 83578101 Saccharomycescerevisiae

As an alternative to the large multienzyme PDH complexes describedabove, some organisms utilize enzymes in the 2-ketoacid oxidoreductasefamily (OFOR) to catalyze acylating oxidative decarboxylation of2-keto-acids. Unlike the PDH complexes, PFOR enzymes contain iron-sulfurclusters, utilize different cofactors and use ferredoxin or flavodixinas electron acceptors in lieu of NAD(P)H. Pyruvate ferredoxinoxidoreductase (PFOR) can catalyze the oxidation of pyruvate to formacetyl-CoA (FIG. 2H). The PFOR from Desulfovibrio africanus has beencloned and expressed in E. coli resulting in an active recombinantenzyme that was stable for several days in the presence of oxygen(Pieulle et al., J Bacteriol. 179:5684-5692 (1997)). Oxygen stability isrelatively uncommon in PFORs and is believed to be conferred by a 60residue extension in the polypeptide chain of the D. africanus enzyme.The M. thermoacetica PFOR is also well characterized (Menon et al.,Biochemistry 36:8484-8494 (1997)) and was even shown to have highactivity in the direction of pyruvate synthesis during autotrophicgrowth (Furdui et al., J Biol Chem. 275:28494-28499 (2000)). Further, E.coli possesses an uncharacterized open reading frame, ydbK, that encodesa protein that is 51% identical to the M. thermoacetica PFOR. Evidencefor pyruvate oxidoreductase activity in E. coli has been described(Blaschkowski et al., Eur. J Biochem. 123:563-569 (1982)). Severaladditional PFOR enzymes are described in Ragsdale, Chem. Rev.103:2333-2346 (2003). Finally, flavodoxin reductases (e.g., fqrB fromHelicobacter pylori or Campylobacter jejuni (St Maurice et al., J.Bacteriol. 189:4764-4773 (2007))) or Rnf-type proteins (Seedorf et al.,Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008); Herrmann et al., J.Bacteriol. 190:784-791 (2008)) provide a means to generate NADH or NADPHfrom the reduced ferredoxin generated by PFOR. These proteins areidentified below.

Protein GenBank ID GI Number Organism Por CAA70873.1 1770208Desulfovibrio africanus Por YP_428946.1 83588937 Moorella thermoaceticaydbK NP_415896.1 16129339 Escherichia coli fqrB NP_207955.1 15645778Helicobacter pylori fqrB YP_001482096.1 157414840 Campylobacter jejuniRnfC EDK33306.1 146346770 Clostridium kluyveri RnfD EDK33307.1 146346771Clostridium kluyveri RnfG EDK33308.1 146346772 Clostridium kluyveri RnfEEDK33309.1 146346773 Clostridium kluyveri RnfA EDK33310.1 146346774Clostridium kluyveri RnfB EDK33311.1 146346775 Clostridium kluyveri

Pyruvate formate-lyase (PFL, EC 2.3.1.54) (FIG. 2H), encoded by pflB inE. coli, can convert pyruvate into acetyl-CoA and formate. The activityof PFL can be enhanced by an activating enzyme encoded by pflA (Knappeet al., Proc. Natl. Acad. Sci U.S.A 81:1332-1335 (1984); Wong et al.,Biochemistry 32:14102-14110 (1993)). Keto-acid formate-lyase (EC2.3.1.-), also known as 2-ketobutyrate formate-lyase (KFL) and pyruvateformate-lyase 4, is the gene product of tdcE in E. coli. This enzymecatalyzes the conversion of 2-ketobutyrate to propionyl-CoA and formateduring anaerobic threonine degradation, and can also substitute forpyruvate formate-lyase in anaerobic catabolism (Simanshu et al., JBiosci. 32:1195-1206 (2007)). The enzyme is oxygen-sensitive and, likePflB, can require post-translational modification by PFL-AE to activatea glycyl radical in the active site (Hesslinger et al., Mol. Microbiol27:477-492 (1998)). A pyruvate formate-lyase from Archaeoglobus fulgidusencoded by pflD has been cloned, expressed in E. coli and characterized(Lehtio et al., Protein Eng Des Sel 17:545-552 (2004)). The crystalstructures of the A. fulgidus and E. coli enzymes have been resolved(Lehtio et al., J Mol. Biol. 357:221-235 (2006); Leppanen et al.,Structure. 7:733-744 (1999)). Additional PFL and PFL-AE candidates arefound in Lactococcus lactis (Melchiorsen et al., Appl MicrobiolBiotechnol 58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbeet al., Oral. Microbiol Immunol. 18:293-297 (2003)), Chlamydomonasreinhardtii (Hemschemeier et al., Eukaryot. Cell 7:518-526 (2008b);Atteia et al., J. Biol. Chem. 281:9909-9918 (2006)) and Clostridiumpasteurianum (Weidner et al., J Bacteriol. 178:2440-2444 (1996)).

Protein GenBank ID GI Number Organism pflB NP_415423 16128870Escherichia coli pflA NP_415422.1 16128869 Escherichia coli tdcEAAT48170.1 48994926 Escherichia coli pflD NP_070278.1 11499044Archaeoglobus fulgidus pfl CAA03993 2407931 Lactococcus lactis pflBAA09085 1129082 Streptococcus mutans PFL1 XP_001689719.1 159462978Chlamydomonas reinhardtii pflA1 XP_001700657.1 159485246 Chlamydomonasreinhardtii pfl Q46266.1 2500058 Clostridium pasteurianum act CAA63749.11072362 Clostridium pasteurianum

If a pyruvate formate lyase is utilized to convert pyruvate toacetyl-CoA, coexpression of a formate dehydrogenase or formate hydrogenlyase enzyme will converte formate to carbon dioxide. Formatedehydrogenase (FDH) catalyzes the reversible transfer of electrons fromformate to an acceptor. Enzymes with FDH activity utilize variouselectron carriers such as, for example, NADH (EC 1.2.1.2), NADPH (EC1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) andhydrogenases (EC 1.1.99.33). FDH enzymes have been characterized fromMoorella thermoacetica (Andreesen and Ljungdahl, J Bacteriol 116:867-873(1973); Li et al., J Bacteriol 92:405-412 (1966); Yamamoto et al., JBiol Chem. 258:1826-1832 (1983). The loci, Moth 2312 is responsible forencoding the alpha subunit of formate dehydrogenase while the betasubunit is encoded by Moth_(—)2314 (Pierce et al., Environ Microbiol(2008)). Another set of genes encoding formate dehydrogenase activitywith a propensity for CO₂ reduction is encoded by Sfum_(—)2703 throughSfum_(—)2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur JBiochem. 270:2476-2485 (2003)); Reda et al., PNAS 105:10654-10658(2008)). A similar set of genes presumed to carry out the same functionare encoded by CHY_(—)0731, CHY_(—)0732, and CHY_(—)0733 in C.hydrogenoformans (Wu et al., PLoS Genet 1:e65 (2005)). Formatedehydrogenases are also found many additional organisms including C.carboxidivorans P7, Bacillus methanolicus, Burkholderia stabilis,Moorella thermoacetica ATCC 39073, Candida boidinii, Candida methylica,and Saccharomyces cerevisiae S288c.

Protein GenBank ID GI Number Organism Moth_2312 YP_431142 148283121Moorella thermoacetica Moth_2314 YP_431144 83591135 Moorellathermoacetica Sfum_2703 YP_846816.1 116750129 Syntrophobacterfumaroxidans Sfum 2704 YP_846817.1 116750130 Syntrophobacterfumaroxidans Sfum 2705 YP_846818.1 116750131 Syntrophobacterfumaroxidans Sfum 2706 YP_846819.1 116750132 Syntrophobacterfumaroxidans CHY_0731 YP_359585.1 78044572 Carboxydothermushydrogenoformans CHY_0732 YP_359586.1 78044500 Carboxydothermushydrogenoformans CHY_0733 YP_359587.1 78044647 Carboxydothermushydrogenoformans CcarbDRAFT_0901 ZP_05390901.1 255523938 Clostridiumcarboxidivorans P7 CcarbDRAFT_4380 ZP_05394380.1 255527512 Clostridiumcarboxidivorans P7 fdhA, MGA3_06625 EIJ82879.1 387590560 Bacillusmethanolicus MGA3 fdhA, PB1_11719 ZP_10131761.1 387929084 Bacillusmethanolicus PB1 fdhD, MGA3_06630 EIJ82880.1 387590561 Bacillusmethanolicus MGA3 fdhD, PB1_11724 ZP_10131762.1 387929085 Bacillusmethanolicus PB1 fdh ACF35003. 194220249 Burkholderia stabilis FDH1AAC49766.1 2276465 Candida boidinii fdh CAA57036.1 1181204 Candidamethylica FDH2 P0CF35.1 294956522 Saccharomyces cerevisiae S288c FDH1NP_015033.1 6324964 Saccharomyces cerevisiae S288c

Alternately, a formate hydrogen lyase enzyme can be employed to convertformate to carbon dioxide and hydrogen. An exemplary formate hydrogenlyase enzyme can be found in Escherichia coli. The E. coli formatehydrogen lyase consists of hydrogenase 3 and formate dehydrogenase-H(Maeda et al., Appl Microbiol Biotechnol 77:879-890 (2007)). It isactivated by the gene product of fhlA. (Maeda et al., Appl MicrobiolBiotechnol 77:879-890 (2007)). The addition of the trace elements,selenium, nickel and molybdenum, to a fermentation broth has been shownto enhance formate hydrogen lyase activity (Soini et al., Microb. CellFact. 7:26 (2008)). Various hydrogenase 3, formate dehydrogenase andtranscriptional activator genes are shown below. A formate hydrogenlyase enzyme also exists in the hyperthermophilic archaeon, Thermococcuslitoralis (Takacs et al., BMC. Microbiol 8:88 (2008)). Additionalformate hydrogen lyase systems have been found in Salmonellatyphimurium, Klebsiella pneumoniae, Rhodospirillum rubrum,Methanobacterium formicicum (Vardar-Schara et al., MicrobialBiotechnology 1:107-125 (2008)).

Protein GenBank ID GI number Organism hycA NP_417205 16130632Escherichia coli K-12 MG1655 hycB NP_417204 16130631 Escherichia coliK-12 MG1655 hycC NP_417203 16130630 Escherichia coli K-12 MG1655 hycDNP_417202 16130629 Escherichia coli K-12 MG1655 hycE NP_417201 16130628Escherichia coli K-12 MG1655 hycF NP_417200 16130627 Escherichia coliK-12 MG1655 hycG NP_417199 16130626 Escherichia coli K-12 MG1655 hycHNP_417198 16130625 Escherichia coli K-12 MG1655 hycI NP_417197 16130624Escherichia coli K-12 MG1655 fdhF NP_418503 16131905 Escherichia coliK-12 MG1655 fhlA NP_417211 16130638 Escherichia coli K-12 MG1655 mhyCABW05543 157954626 Thermococcus litoralis mhyD ABW05544 157954627Thermococcus litoralis mhyE ABW05545 157954628 Thermococcus litoralismyhF ABW05546 157954629 Thermococcus litoralis myhG ABW05547 157954630Thermococcus litoralis myhH ABW05548 157954631 Thermococcus litoralisfdhA AAB94932 2746736 Thermococcus litoralis fdhB AAB94931 157954625Thermococcus litoralis

Pyruvate:NADP oxidoreductase (PNO) catalyzes the conversion of pyruvateto acetyl-CoA. This enzyme is encoded by a single gene and the activeenzyme is a homodimer, in contrast to the multi-subunit PDH enzymecomplexes described above. The enzyme from Euglena gracilis isstabilized by its cofactor, thiamin pyrophosphate (Nakazawa et al, ArchBiochem Biophys 411:183-8 (2003)). The mitochondrial targeting sequenceof this enzyme should be removed for expression in the cytosol. The PNOprotein of E. gracilis protein and other NADP-dependant pyruvate:NADP+oxidoreductase enzymes are listed in the table below.

Protein GenBank ID GI Number Organism PNO Q94IN5.1 33112418 Euglenagracilis cgd4_690 XP_625673.1 66356990 Cryptosporidium parvum Iowa IITPP_PFOR_PNO XP_002765111.11 294867463 Perkinsus marinus ATCC 50983

The NAD(P)⁺ dependent oxidation of acetaldehyde to acetyl-CoA (FIG. 2I)can be catalyzed by an acylating acetaldehyde dehydrogenase (EC1.2.1.10). Acylating acetaldehyde dehydrogenase enzymes of E. coli areencoded by adhE, eutE, and mhpF (Ferrandez et al, J Bacteriol179:2573-81 (1997)). The Pseudomonas sp. CF600 enzyme, encoded by dmpF,participates in meta-cleavage pathways and forms a complex with4-hydroxy-2-oxovalerate aldolase (Shingler et al, J Bacteriol 174:711-24(1992)). Solventogenic organisms such as Clostridium acetobutylicumencode bifunctional enzymes with alcohol dehydrogenase and acetaldehydedehydrogenase activities. The bifunctional C. acetobutylicum enzymes areencoded by bdh I and adhE2 (Walter, et al., J. Bacteriol. 174:7149-7158(1992); Fontaine et al., J. Bacteriol. 184:821-830 (2002)). Yet anothercandidate for acylating acetaldehyde dehydrogenase is the ald gene fromClostridium beijerinckii (Toth, Appl. Environ. Microbiol. 65:4973-4980(1999). This gene is very similar to the eutE acetaldehyde dehydrogenasegenes of Salmonella typhimurium and E. coli (Toth, Appl. Environ.Microbiol. 65:4973-4980 (1999).

Protein GenBank ID GI Number Organism adhE NP_415757.1 16129202Escherichia coli mhpF NP_414885.1 16128336 Escherichia coli dmpFCAA43226.1 45683 Pseudomonas sp. CF600 adhE2 AAK09379.1 12958626Clostridium acetobutylicum bdh I NP_349892.1 15896543 Clostridiumacetobutylicum Ald AAT66436 49473535 Clostridium beijerinckii eutENP_416950 16130380 Escherichia coli eutE AAA80209 687645 Salmonellatyphimurium

Threonine aldolase (EC 4.1.2.5) catalyzes the cleavage of threonine toglycine and acetaldehyde (FIG. 2J). The Saccharomyces cerevisiae andCandida albicans enzymes are encoded by GLY1 (Liu et al, Eur J Biochem245:289-93 (1997); McNeil et al, Yeast 16:167-75 (2000)). The ltaE andglyA gene products of E. coli also encode enzymes with this activity(Liu et al, Eur J Biochem 255:220-6 (1998)).

Protein GenBank ID GI Number Organism GLY1 NP_010868.1 6320789Saccharomyces cerevisiae GLY1 AAB64198.1 2282060 Candida albicans ltaEAAC73957.1 1787095 Escherichia coli glyA AAC75604.1 1788902 Escherichiacoli

Example III Pathways for Producing Acetyl-CoA from PEP and Pyruvate

Pathways for the conversion of cytosolic phosphoenolpyruvate (PEP) andpyruvate to cytosolic acetyl-CoA can also enable deployment of acytosolic fatty alcohol, fatty aldehyde or fatty acid production pathwayfrom acetyl-CoA. FIG. 3 shows numerous pathways for converting PEP andpyruvate to acetyl-CoA.

The conversion of PEP to oxaloacetate is catalyzed in one, two or threeenzymatic steps. Oxaloacetate is further converted to acetyl-CoA viamalonate semialdehyde or malonyl-CoA intermediates. In one pathway, PEPcarboxylase or PEP carboxykinase converts PEP to oxaloacetate (step A);oxaloacetate decarboxylase converts the oxaloacetate to malonate (stepB); and malonate semialdehyde dehydrogenase (acetylating) converts themalonate semialdehyde to acetyl-CoA (step C). In another pathwaypyruvate kinase or PEP phosphatase converts PEP to pyruvate (step N);pyruvate carboxylase converts the pyruvate to (step H); oxaloacetatedecarboxylase converts the oxaloacetate to malonate (step B); andmalonate semialdehyde dehydrogenase (acetylating) converts the malonatesemialdehyde to acetyl-CoA (step C). In another pathway pyruvate kinaseor PEP phosphatase converts PEP to pyruvate (step N); malic enzymeconverts the pyruvate to malate (step L); malate dehydrogenase oroxidoreductase converts the malate to oxaloacetate (step M);oxaloacetate decarboxylase converts the oxaloacetate to malonate (stepB); and malonate semialdehyde dehydrogenase (acetylating) converts themalonate semialdehyde to acetyl-CoA (step C). In another pathway, PEPcarboxylase or PEP carboxykinase converts PEP to oxaloacetate (step A);oxaloacetate decarboxylase converts the oxaloacetate to malonatesemialdehyde (step B); malonyl-CoA reductase converts the malonatesemialdehyde to malonyl-CoA (step G); and malonyl-CoA decarboxylaseconverts the malonyl-CoA to acetyl-CoA (step (D). In another pathway,pyruvate kinase or PEP phosphatase converts PEP to pyruvate (step N);pyruvate carboxylase converts the pyruvate to oxaloacetate (step H);(oxaloacetate decarboxylase converts the oxaloacetate to malonatesemialdehyde (step B); malonyl-CoA reductase converts the malonatesemialdehyde to malonyl-CoA (step G); and malonyl-CoA decarboxylaseconverts the malonyl-CoA to acetyl-CoA (step (D). In another pathway,pyruvate kinase or PEP phosphatase converts PEP to pyruvate (step N);malic enzyme converts the pyruvate to malate (step L); malatedehydrogenase or oxidoreductase converts the malate to oxaloacetate(step M); oxaloacetate decarboxylase converts the oxaloacetate tomalonate semialdehyde (step B); malonyl-CoA reductase converts themalonate semialdehyde to malonyl-CoA (step G); and malonyl-CoAdecarboxylase converts the malonyl-CoA to acetyl-CoA (step (D). Inanother pathway, PEP carboxylase or PEP carboxykinase converts PEP tooxaloacetate (step A); oxaloacetate decarboxylase converts theoxaloacetate to malonate semialdehyde (step B); malonate semialdehydedehydrogenase converts the malonate semialdehyde to malonate (step J);malonyl-CoA synthetase or transferase converts the malonate tomalonyl-CoA (step K); and malonyl-CoA decarboxylase converts themalonyl-CoA to acetyl-CoA (step D). In another pathway, pyruvate kinaseor PEP phosphatase converts PEP to pyruvate (step N); pyruvatecarboxylase converts the pyruvate to oxaloacetate (step H); oxaloacetatedecarboxylase converts the oxaloacetate to malonate semialdehyde (stepB); malonate semialdehyde dehydrogenase converts the malonatesemialdehyde to malonate (step J); malonyl-CoA synthetase or transferaseconverts the malonate to malonyl-CoA (step K); and malonyl-CoAdecarboxylase converts the malonyl-CoA to acetyl-CoA (step D). Inanother pathway, pyruvate kinase or PEP phosphatase converts PEP topyruvate (step N); malic enzyme converts the pyruvate to malate (stepL); malate dehydrogenase or oxidoreductase converts the malate tooxaloacetate (step M); oxaloacetate decarboxylase converts theoxaloacetate to malonate semialdehyde (step B); malonate semialdehydedehydrogenase converts the malonate semialdehyde to malonate (step J);malonyl-CoA synthetase or transferase converts the malonate tomalonyl-CoA (step K); and malonyl-CoA decarboxylase converts themalonyl-CoA to acetyl-CoA (step D). In another pathway, PEP carboxylaseor PEP carboxykinase converts PEP to oxaloacetate (step A); oxaloacetatedehydrogenase or oxaloacetate oxidoreductase converts the oxaloacetateto malonyl-CoA (step F); and malonyl-CoA decarboxylase converts themalonyl-CoA to acetyl-CoA (step D). In another pathway, pyruvate kinaseor PEP phosphatase converts PEP to pyruvate (step N); pyruvatecarboxylase converts the pyruvate to oxaloacetate (step H); oxaloacetatedehydrogenase or oxaloacetate oxidoreductase converts the oxaloacetateto malonyl-CoA (step F); and malonyl-CoA decarboxylase converts themalonyl-CoA to acetyl-CoA (step D). In another pathway, pyruvate kinaseor PEP phosphatase converts PEP to pyruvate (step N); malic enzymeconverts the pyruvate to malate (step L); malate dehydrogenase oroxidoreductase converts the malate to oxaloacetate (step M);oxaloacetate dehydrogenase or oxaloacetate oxidoreductase converts theoxaloacetate to malonyl-CoA (step F); and malonyl-CoA decarboxylaseconverts the malonyl-CoA to acetyl-CoA (step D).

Enzymes candidates for the reactions shown in FIG. 3 are describedbelow.

1.1.n.a Oxidoreductase (alcohol to oxo) M 1.1.1.d Malic enzyme L 1.2.1.aOxidoreductase (aldehyde to acid) J 1.2.1.b Oxidoreductase (acyl-CoA toaldehyde) G 1.2.1.f Oxidoreductase (decarboxylating acyl-CoA to Caldehyde) 2.7.2.a Kinase N 2.8.3.a CoA transferase K 3.1.3.a PhosphataseN 4.1.1.a Decarboxylase A, B, D 6.2.1.a CoA synthetase K 6.4.1.aCarboxylase D, H

Enzyme candidates for several enzymes in FIG. 3 have been describedelsewhere herein. These include acetyl-CoA carboxylase, acetoacetyl-CoAsynthase, acetoacetyl-CoA thiolase, malonyl-CoA reductase (also calledmalonate semialdehyde dehydrogenase (acylating), malate dehydrogenase.

1.1.n.a Oxidoreductase (Alcohol to Oxo)

Malate dehydrogenase or oxidoreductase catalyzes the oxidation of malateto oxaloacetate. Different carriers can act as electron acceptors forenzymes in this class. Malate dehydrogenase enzymes utilize NADP or NADas electron acceptors. Malate dehydrogenase (Step M) enzyme candidatesare described above in example 1 (Table 7, 23). Malate:quinoneoxidoreductase enzymes (EC 1.1.5.4) are membrane-associated and utilizequinones, flavoproteins or vitamin K as electron acceptors.Malate:quinone oxidoreductase enzymes of E. coli, Helicobacter pyloriand Pseudomonas syringae are encoded by mqo (Kather et al, J Bacteriol182:3204-9 (2000); Mellgren et al, J Bacteriol 191:3132-42 (2009)). TheCgl2001 gene of C. gluamicum also encodes an MQO enzyme (Mitsuhashi etal, Biosci Biotechnol Biochem 70:2803-6 (2006)).

Protein GenBank ID GI Number Organism mqo NP_416714.1 16130147Escherichia coli mqo NP_206886.1 15644716 Helicobacter pylori mqoNP_790970.1 28868351 Pseudomonas syringae Cgl2001 NP_601207.1 19553205Corynebacterium glutamicum

1.1.1.d Malic Enzyme

Malic enzyme (malate dehydrogenase) catalyzes the reversible oxidativecarboxylation of pyruvate to malate. E. coli encodes two malic enzymes,MaeA and MaeB (Takeo, J. Biochem. 66:379-387 (1969)). Although malicenzyme is typically assumed to operate in the direction of pyruvateformation from malate, the NAD-dependent enzyme, encoded by maeA, hasbeen demonstrated to operate in the carbon-fixing direction (Stols andDonnelly, Appl. Environ. Microbiol. 63(7) 2695-2701 (1997)). A similarobservation was made upon overexpressing the malic enzyme from Ascarissuum in E. coli (Stols et al., Appl. Biochem. Biotechnol. 63-65(1),153-158 (1997)). The second E. coli malic enzyme, encoded by maeB, isNADP-dependent and also decarboxylates oxaloacetate and other alpha-ketoacids (Iwakura et al., J. Biochem. 85(5):1355-65 (1979)). Anothersuitable enzyme candidate is me1 from Zea mays (Furumoto et al, PlantCell Physiol 41:1200-1209 (2000)).

Protein GenBank ID GI Number Organism maeA NP_415996 90111281Escherichia coli maeB NP_416958 16130388 Escherichia coli NAD-ME P27443126732 Ascaris suum Me1 P16243.1 126737 Zea mays

1.2.1.a Oxidoreductase (Aldehyde to Acid)

The oxidation of malonate semialdehyde to malonate is catalyzed bymalonate semialdehyde dehydrogenase (EC 1.2.1.15). This enzyme wascharacterized in Pseudomonas aeruginosa (Nakamura et al, Biochim BiophysActa 50:147-52 (1961)). The NADP and NAD-dependent succinatesemialdehyde dehydrogenase enzymes of Euglena gracilas accept malonatesemialdehyde as substrates (Tokunaga et al, Biochem Biophys Act429:55-62 (1976)). Genes encoding these enzymes has not been identifiedto date. Aldehyde dehydrogenase enzymes from eukoryotic organisms suchas S. cerevisiae, C. albicans, Y. lipolytica and A. niger typically havebroad substrate specificity and are suitable candidates. These enzymesand other acid forming aldehyde dehydrogenase and aldehyde oxidaseenzymes are described earlier and listed in Tables 9 and 30. AdditionalMSA dehydrogenase enzyme candidates include NAD(P)+-dependent aldehydedehydrogenase enzymes (EC 1.2.1.3). Two aldehyde dehydrogenases found inhuman liver, ALDH-1 and ALDH-2, have broad substrate ranges for avariety of aliphatic, aromatic and polycyclic aldehydes (Klyosov,Biochemistry 35:4457-4467 (1996a)). Active ALDH-2 has been efficientlyexpressed in E. coli using the GroEL proteins as chaperonins (Lee etal., Biochem. Biophys. Res. Commun. 298:216-224 (2002)). The ratmitochondrial aldehyde dehydrogenase also has a broad substrate range(Siew et al., Arch. Biochem. Biophys. 176:638-649 (1976)). The E. coligenes astD and aldH encode NAD+-dependent aldehyde dehydrogenases. AstDis active on succinic semialdehyde (Kuznetsova et al., FEMS MicrobiolRev 29:263-279 (2005)) and aldH is active on a broad range of aromaticand aliphatic substrates (Jo et al, Appl Microbiol Biotechnol 81:51-60(2008)).

GenBank Gene Accession No. GI No. Organism astD P76217.1 3913108Escherichia coli aldH AAC74382.1 1787558 Escherichia coli ALDH-2P05091.2 118504 Homo sapiens ALDH-2 NP_115792.1 14192933 Rattusnorvegicus

1.2.1.f Oxidoreductase (Decarboxylating Acyl-CoA to Aldehyde)

Malonate semialdehyde dehydrogenase (acetylating) (EC 1.2.1.18)catalyzes the oxidative decarboxylation of malonate semialdehyde toacetyl-CoA. Exemplary enzymes are encoded by ddcC of Halomonas sp. HTNK1(Todd et al, Environ Microbiol 12:237-43 (2010)) and IolA ofLactobacillus casei (Yebra et al, AEM 73:3850-8 (2007)). The DdcC enzymehas homologs in A. niger and C. albicans, shown in the table below. Themalonate semialdehyde dehydrogenase enzyme in Rattus norvegicus, Mmsdh,also converts malonate semialdehyde to acetyl-CoA (U.S. Pat. No.8,048,624). A malonate semialdehyde dehydrogenase (acetylating) enzymehas also been characterized in Pseudomonas fluorescens, although thegene has not been identified to date (Hayaishi et al, J Biol Chem236:781-90 (1961)). Methylmalonate semialdehyde dehydrogenase(acetylating) enzymes (EC 1.2.1.27) are also suitable candidates, asseveral enzymes in this class accept malonate semialdehyde as asubstrate including Msdh of Bacillus subtilis (Stines-Chaumeil et al,Biochem J 395:107-15 (2006)) and the methylmalonate semialdehydedehydrogenase of R. norvegicus (Kedishvii et al, Methods Enzymol324:207-18 (2000)).

Protein GenBank ID GI Number Organism ddcC ACV84070.1 258618587Halomonas sp. HTNK1 ANI_1_1120014 XP_001389265.1 145229913 Aspergillusniger ALD6 XP_710976.1 68490403 Candida albicans YALI0C01859gXP_501343.1 50547747 Yarrowia lipolytica mmsA_1 YP_257876.1 70734236Pseudomonas fluorescens mmsA_2 YP_257884.1 70734244 Pseudomonasfluorescens PA0130 NP_248820.1 15595328 Pseudomonas aeruginosa MmsdhQ02253.1 400269 Rattus norvegicus msdh NP_391855.1 16081027 Bacillussubtilis IolA ABP57762.1 145309085 Lactobacillus casei

2.7.2.a Kinase

Pyruvate kinase (Step 10N), also known as phosphoenolpyruvate synthase(EC 2.7.9.2), converts pyruvate and ATP to PEP and AMP. This enzyme isencoded by the PYK1 (Burke et al., J. Biol. Chem. 258:2193-2201 (1983))and PYK2 (Boles et al., J. Bacteriol. 179:2987-2993 (1997)) genes in S.cerevisiae. In E. coli, this activity is catalyzed by the gene productsof pykF and pykA. Selected homologs of the S. cerevisiae enzymes arealso shown in the table below.

Protein GenBank ID GI Number Organism PYK1 NP_009362 6319279Saccharomyces cerevisiae PYK2 NP_014992 6324923 Saccharomyces cerevisiaepykF NP_416191.1 16129632 Escherichia coli pykA NP_416368.1 16129807Escherichia coli KLLA0F23397g XP_456122.1 50312181 Kluyveromyces lactisCaO19.3575 XP_714934.1 68482353 Candida albicans CaO19.11059 XP_714997.168482226 Candida albicans YALI0F09185p XP_505195 210075987 Yarrowialipolytica ANI_1_1126064 XP_001391973 145238652 Apergillus niger

2.8.3.a CoA Transferase

Activation of malonate to malonyl-CoA is catalyzed by a CoA transferasein EC class 2.8.3.a. Malonyl-CoA:acetate CoA transferase (EC 2.8.3.3)enzymes have been characterized in Pseudomonas species includingPseudomonas fluorescens and Pseudomonas putida (Takamura et al, BiochemInt 3:483-91 (1981); Hayaishi et al, J Biol Chem 215:125-36 (1955)).Genes associated with these enzymes have not been identified to date. Amitochondrial CoA transferase found in Rattus norvegicus liver alsocatalyzes this reaction and is able to utilize a range of CoA donors andacceptors (Deana et al, Biochem Int 26:767-73 (1992)). Several CoAtransferase enzymes described above can also be applied to catalyze stepK of FIG. 10. These enzymes include acetyl-CoA transferase (Table 26),3-HB CoA transferase (Table 8), acetoacetyl-CoA transferase (table 55),SCOT (table 56) and other CoA transferases (table 57).

3.1.3.a Phosphatase

Phosphoenolpyruvate phosphatase (EC 3.1.3.60, Step 10N) catalyzes thehydrolysis of PEP to pyruvate and phosphate. Numerous phosphataseenzymes catalyze this activity, including alkaline phosphatase (EC3.1.3.1), acid phosphatase (EC 3.1.3.2), phosphoglycerate phosphatase(EC 3.1.3.20) and PEP phosphatase (EC 3.1.3.60). PEP phosphatase enzymeshave been characterized in plants such as Vignia radiate, Bruguierasexangula and Brassica nigra. The phytase from Aspergillus fumigates,the acid phosphatase from Homo sapiens and the alkaline phosphatase ofE. coli also catalyze the hydrolysis of PEP to pyruvate (Brugger et al,Appl Microbiol Biotech 63:383-9 (2004); Hayman et al, Biochem J261:601-9 (1989); et al, The Enzymes 3^(rd) Ed. 4:373-415 (1971))).Similar enzymes have been characterized in Campylobacter jejuni (vanMourik et al., Microbiol. 154:584-92 (2008)), Saccharomyces cerevisiae(Oshima et al., Gene 179:171-7 (1996)) and Staphylococcus aureus (Shahand Blobel, J. Bacteriol. 94:780-1 (1967)). Enzyme engineering and/orremoval of targeting sequences may be required for alkaline phosphataseenzymes to function in the cytoplasm.

Protein GenBank ID GI Number Organism phyA O00092.1 41017447 Aspergillusfumigatus Acp5 P13686.3 56757583 Homo sapiens phoA NP_414917.2 49176017Escherichia coli phoX ZP_01072054.1 86153851 Campylobacter jejuni PHO8AAA34871.1 172164 Saccharomyces cerevisiae SaurJH1_2706 YP_001317815.1150395140 Staphylococcus aureus

4.1.1.a Decarboxylase

Several reactions in FIG. 10 are catalyzed by decarboxylase enzymes inEC class 4.1.1, including oxaloacetate decarboxylase (Step B),malonyl-CoA decarboxylase (step D) and pyruvate carboxylase orcarboxykinase (step A).

Carboxylation of phosphoenolpyruvate to oxaloacetate is catalyzed byphosphoenolpyruvate carboxylase (EC 4.1.1.31). Exemplary PEP carboxylaseenzymes are encoded by ppc in E. coli (Kai et al., Arch. Biochem.Biophys. 414:170-179 (2003), ppcA in Methylobacterium extorquens AM1(Arps et al., J. Bacteriol. 175:3776-3783 (1993), and ppc inCorynebacterium glutamicum (Eikmanns et al., Mol. Gen. Genet.218:330-339 (1989).

Protein GenBank ID GI Number Organism Ppc NP_418391 16131794 Escherichiacoli ppcA AAB58883 28572162 Methylobacterium extorquens Ppc ABB5327080973080 Corynebacterium glutamicum

An alternative enzyme for carboxylating phosphoenolpyruvate tooxaloacetate is PEP carboxykinase (EC 4.1.1.32, 4.1.1.49), whichsimultaneously forms an ATP or GTP. In most organisms PEP carboxykinaseserves a gluconeogenic function and converts oxaloacetate to PEP at theexpense of one ATP. S. cerevisiae is one such organism whose native PEPcarboxykinase, PCK1, serves a gluconeogenic role (Valdes-Hevia et al.,FEBS Lett. 258:313-316 (1989). E. coli is another such organism, as therole of PEP carboxykinase in producing oxaloacetate is believed to beminor when compared to PEP carboxylase (Kim et al., Appl. Environ.Microbiol. 70:1238-1241 (2004)). Nevertheless, activity of the native E.coli PEP carboxykinase from PEP towards oxaloacetate has been recentlydemonstrated in ppc mutants of E. coli K-12 (Kwon et al., J. Microbiol.Biotechnol. 16:1448-1452 (2006)). These strains exhibited no growthdefects and had increased succinate production at high NaHCO₃concentrations. Mutant strains of E. coli can adopt Pck as the dominantCO₂-fixing enzyme following adaptive evolution (Zhang et al. 2009). Insome organisms, particularly rumen bacteria, PEP carboxykinase is quiteefficient in producing oxaloacetate from PEP and generating ATP.Examples of PEP carboxykinase genes that have been cloned into E. coliinclude those from Mannheimia succiniciproducens (Lee et al.,Biotechnol. Bioprocess Eng. 7:95-99 (2002)), Anaerobiospirillumsucciniciproducens (Laivenieks et al., Appl. Environ. Microbiol.63:2273-2280 (1997), and Actinobacillus succinogenes (Kim et al. supra).The PEP carboxykinase enzyme encoded by Haemophilus influenza iseffective at forming oxaloacetate from PEP. Another suitable candidateis the PEPCK enzyme from Megathyrsus maximus, which has a low Km forCO₂, a substrate thought to be rate-limiting in the E. coli enzyme (Chenet al., Plant Physiol 128:160-164 (2002); Cotelesage et al., Int. JBiochem. Cell Biol. 39:1204-1210 (2007)). The kinetics of theGTP-dependent pepck gene product from Cupriavidus necator favoroxaloacetate formation (U.S. Pat. No. 8,048,624 and Lea et al, AminoAcids 20:225-41 (2001)).

Protein GenBank ID GI Number Organism PCK1 NP_013023 6322950Saccharomyces cerevisiae pck NP_417862.1 16131280 Escherichia coli pckAYP_089485.1 52426348 Mannheimia succiniciproducens pckA O09460.1 3122621Anaerobiospirillum succiniciproducens pckA Q6W6X5 75440571Actinobacillus succinogenes pckA P43923.1 1172573 Haemophilus influenzaAF532733.1: AAQ10076.1 33329363 Megathyrsus maximus 1 . . . 1929 pepckYP_728135.1 113869646 Cupriavidus necator

Oxaloacetate decarboxylase catalyzes the decarboxylation of oxaloacetateto malonate semialdehyde. Enzymes catalyzing this reaction include kgdof Mycobacterium tuberculosis (GenBank ID: O50463.4, GI: 160395583).Enzymes evolved from kgd with improved activity and/or substratespecificity for oxaloacetate have also been described (U.S. Pat. No.8,048,624). Additional enzymes useful for catalyzing this reactioninclude keto-acid decarboxylases shown in the table below.

EC number Name 4.1.1.1 Pyruvate decarboxylase 4.1.1.7 Benzoylformatedecarboxylase 4.1.1.40 Hydroxypyruvate decarboxylase 4.1.1.43Ketophenylpyruvate decarboxylase 4.1.1.71 Alpha-ketoglutaratedecarboxylase 4.1.1.72 Branched chain keto-acid decarboxylase 4.1.1.74Indolepyruvate decarboxylase 4.1.1.75 2-Ketoarginine decarboxylase4.1.1.79 Sulfopyruvate decarboxylase 4.1.1.80 Hydroxyphenylpyruvatedecarboxylase 4.1.1.82 Phosphonopyruvate decarboxylase

The decarboxylation of keto-acids is catalyzed by a variety of enzymeswith varied substrate specificities, including pyruvate decarboxylase(EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7),alpha-ketoglutarate decarboxylase and branched-chain alpha-ketoaciddecarboxylase. Pyruvate decarboxylase (PDC), also termed keto-aciddecarboxylase, is a key enzyme in alcoholic fermentation, catalyzing thedecarboxylation of pyruvate to acetaldehyde. The PDC1 enzyme fromSaccharomyces cerevisiae has a broad substrate range for aliphatic2-keto acids including 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvateand 2-phenylpyruvate (22). This enzyme has been extensively studied,engineered for altered activity, and functionally expressed in E. coli(Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001); Li etal., Biochemistry. 38:10004-10012 (1999); ter Schure et al., Appl.Environ. Microbiol. 64:1303-1307 (1998)). The PDC from Zymomonasmobilus, encoded by pdc, also has a broad substrate range and has been asubject of directed engineering studies to alter the affinity fordifferent substrates (Siegert et al., Protein Eng Des Sel 18:345-357(2005)). The crystal structure of this enzyme is available(Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001)). Otherwell-characterized PDC candidates include the enzymes from Acetobacterpasteurians (Chandra et al., 176:443-451 (2001)) and Kluyveromyceslactis (Krieger et al., 269:3256-3263 (2002)).

Protein GenBank ID GI Number Organism pdc P06672.1 118391 Zymomonasmobilis pdc1 P06169 30923172 Saccharomyces cerevisiae pdc Q8L38820385191 Acetobacter pasteurians pdc1 Q12629 52788279 Kluyveromyceslactis

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broadsubstrate range and has been the target of enzyme engineering studies.The enzyme from Pseudomonas putida has been extensively studied andcrystal structures of this enzyme are available (Polovnikova et al.,42:1820-1830 (2003); Hasson et al., 37:9918-9930 (1998)). Site-directedmutagenesis of two residues in the active site of the Pseudomonas putidaenzyme altered the affinity (Km) of naturally and non-naturallyoccurring substrates (Siegert et al., Protein Eng Des Sel 18:345-357(2005)). The properties of this enzyme have been further modified bydirected engineering (Lingen et al., Chembiochem. 4:721-726 (2003);Lingen et al., Protein Eng 15:585-593 (2002)). The enzyme fromPseudomonas aeruginosa, encoded by mdlC, has also been characterizedexperimentally (Barrowman et al., 34:57-60 (1986)). Additional genecandidates from Pseudomonas stutzeri, Pseudomonas fluorescens and otherorganisms can be inferred by sequence homology or identified using agrowth selection system developed in Pseudomonas putida (Henning et al.,Appl. Environ. Microbiol. 72:7510-7517 (2006)).

Protein GenBank ID GI Number Organism mdlC P20906.2 3915757 Pseudomonasputida mdlC Q9HUR2.1 81539678 Pseudomonas aeruginosa dpgB ABN80423.1126202187 Pseudomonas stutzeri ilvB-1 YP_260581.1 70730840 Pseudomonasfluorescens

A third enzyme capable of decarboxylating 2-oxoacids isalpha-ketoglutarate decarboxylase (KGD, EC 4.1.1.71). The substraterange of this class of enzymes has not been studied to date. Anexemplarly KDC is encoded by kad in Mycobacterium tuberculosis (Tian etal., PNAS 102:10670-10675 (2005)). KDC enzyme activity has also beendetected in several species of rhizobia including Bradyrhizobiumjaponicum and Mesorhizobium loti (Green et al., J Bacteriol182:2838-2844 (2000)). Although the KDC-encoding gene(s) have not beenisolated in these organisms, the genome sequences are available andseveral genes in each genome are annotated as putative KDCs. A KDC fromEuglena gracilis has also been characterized but the gene associatedwith this activity has not been identified to date (Shigeoka et al.,Arch. Biochem. Biophys. 288:22-28 (1991)). The first twenty amino acidsstarting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV(Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). Thegene could be identified by testing candidate genes containing thisN-terminal sequence for KDC activity. A novel class of AKG decarboxylaseenzymes has recently been identified in cyanobacteria such asSynechococcus sp. PCC 7002 and homologs (Zhang and Bryant, Science334:1551-3 (2011)).

Protein GenBank ID GI Number Organism kgd O50463.4 160395583Mycobacterium tuberculosis kgd NP_767092.1 27375563 Bradyrhizobiumjaponicum USDA110 kgd NP_105204.1 13473636 Mesorhizobium loti ilvBACB00744.1 169887030 Synechococcus sp. PCC 7002

A fourth candidate enzyme for catalyzing this reaction is branched chainalpha-ketoacid decarboxylase (BCKA). This class of enzyme has been shownto act on a variety of compounds varying in chain length from 3 to 6carbons (Oku et al., J Biol Chem. 263:18386-18396 (1988); Smit et al.,Appl Environ Microbiol 71:303-311 (2005)). The enzyme in Lactococcuslactis has been characterized on a variety of branched and linearsubstrates including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate,3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate (Smitet al., Appl Environ Microbiol 71:303-311 (2005)). The enzyme has beenstructurally characterized (Berg et al., Science. 318:1782-1786 (2007)).Sequence alignments between the Lactococcus lactis enzyme and thepyruvate decarboxylase of Zymomonas mobilus indicate that the catalyticand substrate recognition residues are nearly identical (Siegert et al.,Protein Eng Des Sel 18:345-357 (2005)), so this enzyme would be apromising candidate for directed engineering. Several ketoaciddecarboxylases of Saccharomyces cerevisiae catalyze the decarboxylationof branched substrates, including ARO10, PDC6, PDC5, PDC1 and THI3(Dickenson et al, J Biol Chem 275:10937-42 (2000)). Yet another BCKADenzyme is encoded by rv0853c of Mycobacterium tuberculosis (Werther etal, J Biol Chem 283:5344-54 (2008)). This enzyme is subject toallosteric activation by alpha-ketoacid substrates. Decarboxylation ofalpha-ketoglutarate by a BCKA was detected in Bacillus subtilis;however, this activity was low (5%) relative to activity on otherbranched-chain substrates (Oku and Kaneda, J Biol Chem. 263:18386-18396(1988)) and the gene encoding this enzyme has not been identified todate. Additional BCKA gene candidates can be identified by homology tothe Lactococcus lactis protein sequence. Many of the high-scoring BLASTphits to this enzyme are annotated as indolepyruvate decarboxylases (EC4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme thatcatalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde inplants and plant bacteria. Recombinant branched chain alpha-keto aciddecarboxylase enzymes derived from the E1 subunits of the mitochondrialbranched-chain keto acid dehydrogenase complex from Homo sapiens and Bostaurus have been cloned and functionally expressed in E. coli (Davie etal., J. Biol. Chem. 267:16601-16606 (1992); Wynn et al., J. Biol. Chem.267:12400-12403 (1992); Wynn et al., J. Biol. Chem. 267:1881-1887(1992)). In these studies, the authors found that co-expression ofchaperonins GroEL and GroES enhanced the specific activity of thedecarboxylase by 500-fold (Wynn et al., J. Biol. Chem. 267:12400-12403(1992)). These enzymes are composed of two alpha and two beta subunits.

Protein GenBank ID GI Number Organism kdcA AAS49166.1 44921617Lactococcus lactis PDC6 NP_010366.1 6320286 Saccharomyces cerevisiaePDC5 NP_013235.1 6323163 Saccharomyces cerevisiae PDC1 P06169 30923172Saccharomyces cerevisiae ARO10 NP_010668.1 6320588 Saccharomycescerevisiae THI3 NP_010203.1 6320123 Saccharomyces cerevisiae rv0853cO53865.1 81343167 Mycobacterium tuberculosis BCKDHB NP_898871.1 34101272Homo sapiens BCKDHA NP_000700.1 11386135 Homo sapiens BCKDHB P21839115502434 Bos taurus BCKDHA P11178 129030 Bos taurus

3-Phosphonopyruvate decarboxylase (EC 4.1.1.82) catalyzes thedecarboxylation of 3-phosphonopyruvate to 2-phosphonoacetaldehyde.Exemplary phosphonopyruvate decarboxylase enzymes are encoded by dhpF ofStreptomyces luridus, ppd of Streptomyces viridochromogenes, fom2 ofStreptomyces wedmorensis and bcpC of Streptomyces hygroscopius (Circelloet al, Chem Biol 17:402-11 (2010); Blodgett et al, FEMS Microbiol Lett163:149-57 (2005); Hidaka et al, Mol Gen Genet 249:274-80 (1995);Nakashita et al, Biochim Biophys Acta 1490:159-62 (2000)). TheBacteroides fragilis enzyme, encoded by aepY, also decarboxylatespyruvate and sulfopyruvate (Zhang et al, J Biol Chem 278:41302-8(2003)).

Protein GenBank ID GI Number Organism dhpF ACZ13457.1 268628095Streptomyces luridus Ppd CAJ14045.1 68697716 Streptomycesviridochromogenes Fom2 BAA32496.1 1061008 Streptomyces wedmorensis aepYAAG26466.1 11023509 Bacteroides fragilis

Many oxaloacetate decarboxylase enzymes such as the eda gene product inE. coli (EC 4.1.1.3), act on the terminal acid of oxaloacetate to formpyruvate. Because decarboxylation at the 3-keto acid position competeswith the malonate semialdehyde forming decarboxylation at the2-keto-acid position, this enzyme activity can be knocked out in a hoststrain with a pathway proceeding through a malonate semilaldehydeintermediate.

Malonyl-CoA decarboxylase (EC 4.1.1.9) catalyzes the decarboxylation ofmalonyl-CoA to acetyl-CoA. Enzymes have been characterized in Rhizobiumleguminosarum and Acinetobacter calcoaceticus (An et al, Eur J Biochem257: 395-402 (1998); Koo et al, Eur J Biochem 266:683-90 (1999)).Similar enzymes have been characterized in Streptomyces erythreus(Hunaiti et al, Arch Biochem Biophys 229:426-39 (1984)). A recombinanthuman malonyl-CoA decarboxylase was overexpressed in E. coli (Zhou etal, Prot Expr Pur 34:261-9 (2004)). Methylmalonyl-CoA decarboxylaseenzymes that decarboxylate malonyl-CoA are also suitable candidates. Forexample, the Veillonella parvula enzyme accepts malonyl-CoA as asubstrate (Hilpert et al, Nature 296:584-5 (1982)). The E. coli enzymeis encoded by ygfG (Benning et al., Biochemistry. 39:4630-4639 (2000);Haller et al., Biochemistry. 39:4622-4629 (2000)). The stereospecificity of the E. coli enzyme was not reported, but the enzyme inPropionigenium modestum (Bott et al., Eur. J. Biochem. 250:590-599(1997)) and Veillonella parvula (Huder et al., J. Biol. Chem.268:24564-24571 (1993)) catalyzes the decarboxylation of the(S)-stereoisomer of methylmalonyl-CoA (Hoffmann et al., FEBS. Lett.220:121-125 (1987)). The enzymes from P. modestum and V. parvula arecomprised of multiple subunits that not only decarboxylate(S)-methylmalonyl-CoA, but also create a pump that transports sodiumions across the cell membrane as a means to generate energy.

Protein GenBank ID GI Number Organism YgfG NP_417394 90111512Escherichia coli matA Q9ZIP6 75424899 Rhizobium leguminosarum mdcDAAB97628.1 2804622 Acinetobacter calcoaceticus mdcE AAF20287.1 6642782Acinetobacter calcoaceticus mdcA AAB97627.1 2804621 Acinetobactercalcoaceticus mdcC AAB97630.1 2804624 Acinetobacter calcoaceticus mcdNP_036345.2 110349750 Homo sapiens mmdA CAA05137 2706398 Propionigeniummodestum mmdD CAA05138 2706399 Propionigenium modestum mmdC CAA051392706400 Propionigenium modestum mmdB CAA05140 2706401 Propionigeniummodestum mmdA CAA80872 415915 Veillonella parvula mmdC CAA80873 415916Veillonella parvula mmdE CAA80874 415917 Veillonella parvula mmdDCAA80875 415918 Veillonella parvula mmdB CAA80876 415919 Veillonellaparvula6.2.1.a CoA synthetase

Activation of malonate to malonyl-CoA is catalyzed by a CoA synthetasein EC class 6.2.1.a. CoA synthetase enzymes that catalyze this reactionhave not been described in the literature to date. Several CoAsynthetase enzymes described above can also be applied to catalyze stepK of FIG. 10. These enzymes include acetyl-CoA synthetase (Table 16, 25)and ADP forming CoA synthetases (Table 17).

6.4.1.a Carboxylase

Pyruvate carboxylase (EC 6.4.1.1) converts pyruvate to oxaloacetate atthe cost of one ATP (step H). Exemplary pyruvate carboxylase enzymes areencoded by PYC1 (Walker et al., Biochem. Biophys. Res. Commun.176:1210-1217 (1991) and PYC2 (Walker et al., supra) in Saccharomycescerevisiae, and pyc in Mycobacterium smegmatis (Mukhopadhyay andPurwantini, Biochim. Biophys. Acta 1475:191-206 (2000)).

Protein GenBank ID GI Number Organism PYC1 NP_011453 6321376Saccharomyces cerevisiae PYC2 NP_009777 6319695 Saccharomyces cerevisiaePyc YP_890857.1 118470447 Mycobacterium smegmatis

Example IV Pathways for Producing Cytosolic Acetyl-CoA fromMitochondrial Acetyl-CoA

A mechanism for transporting acetyl-CoA from the mitochondrion to thecytosol can facilitate deployment of a cytosolic fatty alcohol, fattyaldehyde or fatty acid production pathway that originates fromacetyl-CoA. Exemplary mechanisms for exporting acetyl-CoA include thosedepicted in FIGS. 4 and 5, which can involve forming citrate fromacetyl-CoA and oxaloacetate in the mitochondrion, exporting the citratefrom the mitochondrion to the cytosol, and converting the citrate tooxaloacetate and either acetate or acetyl-CoA. In certain embodiments,provided herein are methods for engineering a eukaryotic organism toincrease its availability of cytosolic acetyl-CoA by introducing enzymescapable of carrying out the transformations depicted in any one of FIGS.4 and 5. Exemplary enzymes capable of carrying out the requiredtransformations are also disclosed herein.

The production of cytosolic acetyl-CoA from mitochondrial acetyl-CoA canbe accomplished by a number of pathways, for example, in three to fiveenzymatic steps. In one exemplary pathway, mitochondrial acetyl-CoA andoxaloacetate are combined into citrate by a citrate synthase and thecitrate is exported out of the mitochondrion by a citrate orcitrate/oxaloacetate transporter. Enzymatic conversion of the citrate inthe cytosol results in cytosolic acetyl-CoA and oxaloacetate. Thecytosolic oxaloacetate can then optionally be transported back into themitochondrion by an oxaloacetate transporter and/or acitrate/oxaloacetate transporter. In another exemplary pathway, thecytosolic oxaloacetate is first enzymatically converted into malate inthe cytosol and then optionally transferred into the mitochondrion by amalate transporter and/or a malate/citrate transporter. Mitochondrialmalate can then be converted into oxaloacetate with a mitochondrialmalate dehydrogenase.

In yet another exemplary pathway, mitochondrial acetyl-CoA can beconverted to cytosolic acetyl-CoA via a citramalate intermediate. Forexample, mitochondrial acetyl-CoA and pyruvate are converted tocitramalate by citramalate synthase. Citramalate can then be transportedinto the cytosol by a citramalate or dicarboxylic acid transporter.Cytosolic acetyl-CoA and pyruvate are then regenerated from citramalate,directly or indirectly, and the pyruvate can re-enter the mitochondria.

Along these lines, several exemplary acetyl-CoA pathways for theproduction of cytosolic acetyl-CoA from mitochondrial acetyl-CoA areshown in FIGS. 4 and 5. In one embodiment, mitochondrial oxaloacetate iscombined with mitochondrial acetyl-CoA to form citrate by a citratesynthase. The citrate is transported outside of the mitochondrion by acitrate transporter, a citrate/oxaloacetate transporter or acitrate/malate transporter. Cytosolic citrate is converted intocytosolic acetyl-CoA and oxaloacetate by an ATP citrate lyase. Inanother pathway, cytosolic citrate is converted into acetate andoxaloacetate by a citrate lyase. Acetate can then be converted intocytosolic acetyl-CoA by an acetyl-CoA synthetase or transferase.Alternatively, acetate can be converted by an acetate kinase to acetylphosphate, and the acetyl phosphate can be converted to cytosolicacetyl-CoA by a phosphotransacetylase. Exemplary enzyme candidates foracetyl-CoA pathway enzymes are described below.

The conversion of oxaloacetate and mitochondrial acetyl-CoA is catalyzedby a citrate synthase (FIGS. 4 and 5, step A). In certain embodiments,the citrate synthase is expressed in a mitochondrion of a non-naturallyoccurring eukaryotic organism provided herein.

Protein GenBank ID GI number Organism CIT1 NP_014398.1 6324328Saccharomyces cerevisiae S288c CIT2 NP_009931.1 6319850 Saccharomycescerevisiae S288c CIT3 NP_015325.1 6325257 Saccharomyces cerevisiae S288cYALI0E02684p XP_503469.1 50551989 Yarrowia lipolytica YALI0E00638pXP_503380.1 50551811 Yarrowia lipolytica ANI_1_876084 XP_001393983.1145242820 Aspergillus niger CBS 513.88 ANI_1_1474074 XP_001393195.2317030721 Aspergillus niger CBS 513.88 ANI_1_2950014 XP_001389414.2317026339 Aspergillus niger CBS 513.88 ANI_1_1226134 XP_001396731.1145250435 Aspergillus niger CBS 513.88 gltA NP_415248.1 16128695Escherichia coli K-12 MG1655

Transport of citrate from the mitochondrion to the cytosol can becarried out by several transport proteins. Such proteins either exportcitrate directly (i.e., citrate transporter, FIGS. 4 and 5, step B) tothe cytosol or export citrate to the cytosol while simultaneouslytransporting a molecule such as malate (i.e., citrate/malatetransporter, FIG. 4, step C) or oxaloacetate (i.e., citrate/oxaloacetatetransporter FIG. 5, step C) from the cytosol into the mitochondrion asshown in FIGS. 4 and 5. Exemplary transport enzymes that carry out thesetransformations are provided in the table below.

Protein GenBank ID GI number Organism CTP1 NP_009850.1 6319768Saccharomyces cerevisiae S288c YALI0F26323p XP_505902.1 50556988Yarrowia lipolytica ATEG_09970 EAU29419.1 114187719 Aspergillus terreusNIH2624 KLLA0E18723g XP_454797.1 50309571 Kluyveromyces lactis NRRLY-1140 CTRG_02320 XP_002548023.1 255726194 Candida tropicalis MYA-3404ANI_1_1474094 XP_001395080.1 145245625 Aspergillus niger CBS 513.88 YHM2NP_013968.1 6323897 Saccharomyces cerevisiae S288c DTC CAC84549.119913113 Arabidopsis thaliana DTC1 CAC84545.1 19913105 Nicotiana tabacumDTC2 CAC84546.1 19913107 Nicotiana tabacum DTC3 CAC84547.1 19913109Nicotiana tabacum DTC4 CAC84548.1 19913111 Nicotiana tabacum DTCAAR06239.1 37964368 Citrus junos

ATP citrate lyase (ACL, EC 2.3.3.8, FIGS. 4 and 5, step D), also calledATP citrate synthase, catalyzes the ATP-dependent cleavage of citrate tooxaloacetate and acetyl-CoA. In certain embodiments, ATP citrate lyaseis expressed in the cytosol of a eukaryotic organism. ACL is an enzymeof the RTCA cycle that has been studied in green sulfur bacteriaChlorobium limicola and Chlorobium tepidum. The alpha(4)beta(4)heteromeric enzyme from Chlorobium limicola was cloned and characterizedin E. coli (Kanao et al., Eur. J. Biochem. 269:3409-3416 (2002). The C.limicola enzyme, encoded by aclAB, is irreversible and activity of theenzyme is regulated by the ratio of ADP/ATP. The Chlorobium tepidum arecombinant ACL from Chlorobium tepidum was also expressed in E. coliand the holoenzyme was reconstituted in vitro, in a study elucidatingthe role of the alpha and beta subunits in the catalytic mechanism (Kimand Tabita, J. Bacteriol. 188:6544-6552 (2006). ACL enzymes have alsobeen identified in Balnearium lithotrophicum, Sulfurihydrogenibiumsubterraneum and other members of the bacterial phylum Aquificae (Hugleret al., Environ. Microbiol. 9:81-92 (2007)). This activity has beenreported in some fungi as well. Exemplary organisms include Sordariamacrospora (Nowrousian et al., Curr. Genet. 37:189-93 (2000)),Aspergillus nidulans and Yarrowia lipolytica (Hynes and Murray,Eukaryotic Cell, July: 1039-1048, (2010), and Aspergillus niger (Meijeret al. J. Ind. Microbiol. Biotechnol. 36:1275-1280 (2009). Othercandidates can be found based on sequence homology. Information relatedto these enzymes is tabulated below.

Protein GenBank ID GI Number Organism aclA BAB21376.1 12407237Chlorobium limicola aclB BAB21375.1 12407235 Chlorobium limicola aclAAAM72321.1 21647054 Chlorobium tepidum aclB AAM72322.1 21647055Chlorobium tepidum aclB ABI50084.1 114055039 Sulfurihydrogenibiumsubterraneum aclA AAX76834.1 62199504 Sulfurimonas denitrificans aclBAAX76835.1 62199506 Sulfurimonas denitrificans acl1 XP_504787.1 50554757Yarrowia lipolytica acl2 XP_503231.1 50551515 Yarrowia lipolyticaSPBC1703.07 NP_596202.1 19112994 Schizosaccharomyces pombe SPAC22A12.16NP_593246.1 19114158 Schizosaccharomyces pombe acl1 CAB76165.1 7160185Sordaria macrospora acl2 CAB76164.1 7160184 Sordaria macrospora aclACBF86850.1 259487849 Aspergillus nidulans aclB CBF86848 259487848Aspergillus nidulans

In some organisms the conversion of citrate to oxaloacetate andacetyl-CoA proceeds through a citryl-CoA intermediate and is catalyzedby two separate enzymes, citryl-CoA synthetase (EC 6.2.1.18) andcitryl-CoA lyase (EC 4.1.3.34) (Aoshima, M., Appl. Microbiol.Biotechnol. 75:249-255 (2007). Citryl-CoA synthetase catalyzes theactivation of citrate to citryl-CoA. The Hydrogenobacter thermophilusenzyme is composed of large and small subunits encoded by ccsA and ccsB,respectively (Aoshima et al., Mol. Micrbiol. 52:751-761 (2004)). Thecitryl-CoA synthetase of Aquifex aeolicus is composed of alpha and betasubunits encoded by sucC1 and sucD1 (Hugler et al., Environ. Microbiol.9:81-92 (2007)). Citryl-CoA lyase splits citryl-CoA into oxaloacetateand acetyl-CoA. This enzyme is a homotrimer encoded by ccl inHydrogenobacter thermophilus (Aoshima et al., Mol. Microbiol. 52:763-770(2004)) and aq_(—)150 in Aquifex aeolicus (Hugler et al., supra (2007)).The genes for this mechanism of converting citrate to oxaloacetate andcitryl-CoA have also been reported recently in Chlorobium tepidum (Eisenet al., PNAS 99(14): 9509-14 (2002)).

Protein GenBank ID GI Number Organism ccsA BAD17844.1 46849514Hydrogenobacter thermophilus ccsB BAD17846.1 46849517 Hydrogenobacterthermophilus sucC1 AAC07285 2983723 Aquifex aeolicus sucD1 AAC076862984152 Aquifex aeolicus ccl BAD17841.1 46849510 Hydrogenobacterthermophilus aq_150 AAC06486 2982866 Aquifex aeolicus CT0380 NP_66128421673219 Chlorobium tepidum CT0269 NP_661173.1 21673108 Chlorobiumtepidum CT1834 AAM73055.1 21647851 Chlorobium tepidum

Citrate lyase (EC 4.1.3.6, FIGS. 4 and 5, step E) catalyzes a series ofreactions resulting in the cleavage of citrate to acetate andoxaloacetate. In certain embodiments, citrate lyase is expressed in thecytosol of a eukaryotic organism. The enzyme is active under anaerobicconditions and is composed of three subunits: an acyl-carrier protein(ACP, gamma), an ACP transferase (alpha), and an acyl lyase (beta).Enzyme activation uses covalent binding and acetylation of an unusualprosthetic group, 2′-(5″-phosphoribosyl)-3-′-dephospho-CoA, which issimilar in structure to acetyl-CoA. Acylation is catalyzed by CitC, acitrate lyase synthetase. Two additional proteins, CitG and CitX, areused to convert the apo enzyme into the active holo enzyme (Schneider etal., Biochemistry 39:9438-9450 (2000)). Wild type E. coli does not havecitrate lyase activity; however, mutants deficient in molybdenumcofactor synthesis have an active citrate lyase (Clark, FEMS Microbiol.Lett. 55:245-249 (1990)). The E. coli enzyme is encoded by citEFD andthe citrate lyase synthetase is encoded by citC (Nilekani and SivaRaman,Biochemistry 22:4657-4663 (1983)). The Leuconostoc mesenteroides citratelyase has been cloned, characterized and expressed in E. coli (Bekal etal., J. Bacteriol. 180:647-654 (1998)). Citrate lyase enzymes have alsobeen identified in enterobacteria that utilize citrate as a carbon andenergy source, including Salmonella typhimurium and Klebsiellapneumoniae (Bott, Arch. Microbiol. 167: 78-88 (1997); Bott and Dimroth,Mol. Microbiol. 14:347-356 (1994)). The aforementioned proteins aretabulated below.

Protein GenBank ID GI Number Organism citF AAC73716.1 1786832Escherichia coli cite AAC73717.2 87081764 Escherichia coli citDAAC73718.1 1786834 Escherichia coli citC AAC73719.2 87081765 Escherichiacoli citG AAC73714.1 1786830 Escherichia coli citX AAC73715.1 1786831Escherichia coli citF CAA71633.1 2842397 Leuconostoc mesenteroides citECAA71632.1 2842396 Leuconostoc mesenteroides citD CAA71635.1 2842395Leuconostoc mesenteroides citC CAA71636.1 3413797 Leuconostocmesenteroides citG CAA71634.1 2842398 Leuconostoc mesenteroides citXCAA71634.1 2842398 Leuconostoc mesenteroides citF NP_459613.1 16763998Salmonella typhimurium citE AAL19573.1 16419133 Salmonella typhimuriumcitD NP_459064.1 16763449 Salmonella typhimurium citC NP_459616.116764001 Salmonella typhimurium citG NP_459611.1 16763996 Salmonellatyphimurium citX NP_459612.1 16763997 Salmonella typhimurium citFCAA56217.1 565619 Klebsiella pneumoniae citE CAA56216.1 565618Klebsiella pneumoniae citD CAA56215.1 565617 Klebsiella pneumoniae citCBAH66541.1 238774045 Klebsiella pneumoniae citG CAA56218.1 565620Klebsiella pneumoniae citX AAL60463.1 18140907 Klebsiella pneumoniae

The acylation of acetate to acetyl-CoA is catalyzed by enzymes withacetyl-CoA synthetase activity (FIGS. 4 and 5, step F). In certainembodiments, acetyl-CoA synthetase is expressed in the cytosol of aeukaryotic organism. Two enzymes that catalyze this reaction areAMP-forming acetyl-CoA synthetase (EC 6.2.1.1) and ADP-formingacetyl-CoA synthetase (EC 6.2.1.13). AMP-forming acetyl-CoA synthetase(ACS) is the predominant enzyme for activation of acetate to acetyl-CoA.Exemplary ACS enzymes are found in E. coli (Brown et al., J. Gen.Microbiol. 102:327-336 (1977)), Ralstonia eutropha (Priefert andSteinbuchel, J. Bacteriol. 174:6590-6599 (1992)), Methanothermobacterthermautotrophicus (Ingram-Smith and Smith, Archaea 2:95-107 (2007)),Salmonella enterica (Gulick et al., Biochemistry 42:2866-2873 (2003))and Saccharomyces cerevisiae (Jogl and Tong, Biochemistry 43:1425-1431(2004)).

Protein GenBank ID GI Number Organism acs AAC77039.1 1790505 Escherichiacoli acoE AAA21945.1 141890 Ralstonia eutropha acs1 ABC87079.1 86169671Methanothermobacter thermautotrophicus acs1 AAL23099.1 16422835Salmonella enterica ACS1 Q01574.2 257050994 Saccharomyces cerevisiae

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is anothercandidate enzyme that couples the conversion of acyl-CoA esters to theircorresponding acids with the concurrent synthesis of ATP. Severalenzymes with broad substrate specificities have been described in theliterature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, wasshown to operate on a variety of linear and branched-chain substratesincluding acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate,butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate,indoleacetate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). Theenzyme from Haloarcula marismortui (annotated as a succinyl-CoAsynthetase) accepts propionate, butyrate, and branched-chain acids(isovalerate and isobutyrate) as substrates, and was shown to operate inthe forward and reverse directions (Brasen et al., Arch. Microbiol.182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophiliccrenarchaeon Pyrobaculum aerophilum showed the broadest substrate rangeof all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA(preferred substrate) and phenylacetyl-CoA (Brasen et al., supra(2004)). The enzymes from A. fulgidus, H. marismortui and P. aerophilumhave all been cloned, functionally expressed, and characterized in E.coli (Musfeldt et al., supra; Brasen et al., supra (2004)). Additionalcandidates include the succinyl-CoA synthetase encoded by sucCD in E.coli (Buck et al., Biochemistry 24:6245-6252 (1985)) and the acyl-CoAligase from Pseudomonas putida (Fernandez-Valverde et al., Appl.Environ. Microbiol. 59:1149-1154 (1993)). Information related to theseproteins and genes is shown below.

Protein GenBank ID GI number Organism AF1211 NP_070039.1 11498810Archaeoglobus fulgidus DSM 4304 AF1983 NP_070807.1 11499565Archaeoglobus fulgidus DSM 4304 scs YP_135572.1 55377722 Haloarculamarismortui ATCC 43049 PAE3250 NP_560604.1 18313937 Pyrobaculumaerophilum str. IM2 sucC NP_415256.1 16128703 Escherichia coli sucDAAC73823.1 1786949 Escherichia coli paaF AAC24333.2 22711873 Pseudomonasputida

An alternative method for adding the CoA moiety to acetate is to apply apair of enzymes such as a phosphate-transferring acyltransferase and anacetate kinase (FIGS. 4 and 5, Step F). This activity enables the netformation of acetyl-CoA with the simultaneous consumption of ATP. Incertain embodiments, phosphotransacetylase is expressed in the cytosolof a eukaryotic organism. An exemplary phosphate-transferringacyltransferase is phosphotransacetylase, encoded by pta. The pta genefrom E. coli encodes an enzyme that can convert acetyl-CoA intoacetyl-phosphate, and vice versa (Suzuki, T. Biochim. Biophys. Acta191:559-569 (1969)). This enzyme can also utilize propionyl-CoA insteadof acetyl-CoA forming propionate in the process (Hesslinger et al. Mol.Microbiol 27:477-492 (1998)). Homologs exist in several other organismsincluding Salmonella enterica and Chlamydomonas reinhardtii.

Protein GenBank ID GI number Organism Pta NP_416800.1 16130232Escherichia coli Pta NP_461280.1 16765665 Salmonella enterica subsp.enterica serovar Typhimurium str. LT2 PAT2 XP_001694504.1 159472743Chlamydomonas reinhardtii PAT1 XP_001691787.1 159467202 Chlamydomonasreinhardtii

An exemplary acetate kinase is the E. coli acetate kinase, encoded byackA (Skarstedt and Silverstein J. Biol. Chem. 251:6775-6783 (1976)).Homologs exist in several other organisms including Salmonella entericaand Chlamydomonas reinhardtii. Information related to these proteins andgenes is shown below:

Protein GenBank ID GI number Organism AckA NP_416799.1 16130231Escherichia coli AckA NP_461279.1 16765664 Salmonella enterica subsp.enterica serovar Typhimurium str. LT2 ACK1 XP_001694505.1 159472745Chlamydomonas reinhardtii ACK2 XP_001691682.1 159466992 Chlamydomonasreinhardtii

In some embodiments, cytosolic oxaloacetate is transported back into amitochondrion by an oxaloacetate transporter. Oxaloacetate transportedback into a mitochondrion can then be used in the acetyl-CoA pathwaysdescribed herein. Transport of oxaloacetate from the cytosol to themitochondrion can be carried out by several transport proteins. Suchproteins either import oxaloacetate directly (i.e., oxaloacetatetransporter) to the mitochondrion or import oxaloacetate to the cytosolwhile simultaneously transporting a molecule such as citrate (i.e.,citrate/oxaloacetate transporter) from the mitochondrion into thecytosol as shown in FIG. 5. Exemplary transport enzymes that carry outthese transformations are provided in the table below.

Protein GenBank ID GI number Organism OAC1 NP_012802.1 6322729Saccharomyces cerevisiae S288c KLLA0B12826g XP_452102.1 50304305Kluyveromyces lactis NRRL Y-1140 YALI0E04048g XP_503525.1 50552101Yarrowia lipolytica CTRG_02239 XP_002547942.1 255726032 Candidatropicalis MYA-3404 DIC1 NP_013452.1 6323381 Saccharomyces cerevisiaeS288c YALI0B03344g XP_500457.1 50545838 Yarrowia lipolytica CTRG_02122XP_002547815.1 255725772 Candida tropicalis MYA-3404 PAS_chr4_0877XP_002494326.1 254574434 Pichia pastoris GS115 DTC CAC84549.1 19913113Arabidopsis thaliana DTC1 CAC84545.1 19913105 Nicotiana tabacum DTC2CAC84546.1 19913107 Nicotiana tabacum DTC3 CAC84547.1 19913109 Nicotianatabacum DTC4 CAC84548.1 19913111 Nicotiana tabacum DTC AAR06239.137964368 Citrus junos

In some embodiments, cytosolic oxaloacetate is first converted to malateby a cytosolic malate dehydrogenase (FIG. 4, step H). Cytosolic malateis transported into a mitochondrion by a malate transporter or acitrate/malate transporter (FIG. 4, step I). Mitochondrial malate isthen converted to oxaloacetate by a mitochondrial malate dehydrogenase(FIG. 4, step J). Mitochondrial oxaloacetate can then be used in theacetyl-CoA pathways described herein. Exemplary examples of each ofthese enzymes are provided below.

Oxaloacetate is converted into malate by malate dehydrogenase (EC1.1.1.37, FIG. 4, step H). When malate is the dicarboxylate transportedfrom the cytosol to mitochondrion, expression of both a cytosolic andmitochondrial version of malate dehydrogenase, e.g., as shown in FIG. 3,can be used. S. cerevisiae possesses three copies of malatedehydrogenase, MDHJ (McAlister-Henn and Thompson, J. Bacteriol.169:5157-5166 (1987), MDH2 (Minard and McAlister-Henn, Mol. Cell. Biol.11:370-380 (1991); Gibson and McAlister-Henn, J. Biol. Chem.278:25628-25636 (2003)), and MDH3 (Steffan and McAlister-Henn, J. Biol.Chem. 267:24708-24715 (1992)), which localize to the mitochondrion,cytosol, and peroxisome, respectively. Close homologs to the cytosolicmalate dehydrogenase, MDH2, from S. cerevisiae are found in severalorganisms including Kluyveromyces lactis and Candida tropicalis. E. coliis also known to have an active malate dehydrogenase encoded by mdh.

Protein GenBank ID GI Number Organism MDH1 NP_012838 6322765Saccharomyces cerevisiae MDH2 NP_014515 116006499 Saccharomycescerevisiae MDH3 NP_010205 6320125 Saccharomyces cerevisiae MdhNP_417703.1 16131126 Escherichia coli KLLA0E07525p XP_454288.1 50308571Kluyveromyces lactis NRRL Y-1140 YALI0D16753g XP_502909.1 50550873Yarrowia lipolytica CTRG_01021 XP_002546239.1 255722609 Candidatropicalis MYA-3404

Transport of malate from the cytosol to the mitochondrion can be carriedout by several transport proteins. Such proteins either import malatedirectly (i.e., malate transporter) to the mitochondrion or importmalate to the cytosol while simultaneously transporting a molecule suchas citrate (i.e., citrate/malate transporter) from the mitochondrioninto the cytosol as shown in FIG. 4. Exemplary transport enzymes thatcarry out these transformations are provided in the table below.

Protein GenBank ID GI number Organism OAC1 NP_012802.1 6322729Saccharomyces cerevisiae S288c KLLA0B12826g XP_452102.1 50304305Kluyveromyces lactis NRRL Y-1140 YALI0E04048g XP_503525.1 50552101Yarrowia lipolytica CTRG_02239 XP_002547942.1 255726032 Candidatropicalis MYA-3404 DIC1 NP_013452.1 6323381 Saccharomyces cerevisiaeS288c YALI0B03344g XP_500457.1 50545838 Yarrowia lipolytica CTRG_02122XP_002547815.1 255725772 Candida tropicalis MYA-3404 PAS_chr4_0877XP_002494326.1 254574434 Pichia pastoris GS115 DTC CAC84549.1 19913113Arabidopsis thaliana DTC1 CAC84545.1 19913105 Nicotiana tabacum DTC2CAC84546.1 19913107 Nicotiana tabacum DTC3 CAC84547.1 19913109 Nicotianatabacum DTC4 CAC84548.1 19913111 Nicotiana tabacum DTC AAR06239.137964368 Citrus junos

Malate can be converted into oxaloacetate by malate dehydrogenase (EC1.1.1.37, FIG. 4, step J). When malate is the dicarboxylate transportedfrom the cytosol to mitochondrion, in certain embodiments, both acytosolic and mitochondrial version of malate dehydrogenase isexpressed, as shown in FIGS. 3 and 4. S. cerevisiae possesses threecopies of malate dehydrogenase, MDH1 (McAlister-Henn and Thompson, J.Bacteriol. 169:5157-5166 (1987), MDH2 (Minard and McAlister-Henn, Mol.Cell. Biol. 11:370-380 (1991); Gibson and McAlister-Henn, J. Biol. Chem.278:25628-25636 (2003)), and MDH3 (Steffan and McAlister-Henn, J. Biol.Chem. 267:24708-24715 (1992)), which localize to the mitochondrion,cytosol, and peroxisome, respectively. Close homologs to themitochondrial malate dehydrogenase, MDH1, from S. cerevisiae are foundin several organisms including Kluyveromyces lactis, Yarrowialipolytica, Candida tropicalis. E. coli is also known to have an activemalate dehydrogenase encoded by mdh.

Protein GenBank ID GI Number Organism MDH1 NP_012838 6322765Saccharomyces cerevisiae MDH2 NP_014515 116006499 Saccharomycescerevisiae MDH3 NP_010205 6320125 Saccharomyces cerevisiae MdhNP_417703.1 16131126 Escherichia coli KLLA0F25960g XP_456236.1 50312405Kluyveromyces lactis NRRL Y-1140 YALI0D16753g XP_502909.1 50550873Yarrowia lipolytica CTRG_00226 XP_002545445.1 255721021 Candidatropicalis MYA-3404

Example V Utilization of Pathway Enzymes with a Preference for NADH

The production of acetyl-CoA from glucose can generate at most fourreducing equivalents in the form of NADH. A straightforward and energyefficient mode of maximizing the yield of reducing equivalents is toemploy the Embden-Meyerhof-Parnas glycolysis pathway (EMP pathway). Inmany carbohydrate utilizing organisms, one NADH molecule is generatedper oxidation of each glyceraldehyde-3-phosphate molecule by means ofglyceraldehyde-3-phosphate dehydrogenase. Given that two molecules ofglyceraldehyde-3-phosphate are generated per molecule of glucosemetabolized via the EMP pathway, two NADH molecules can be obtained fromthe conversion of glucose to pyruvate.

Two additional molecules of NADH can be generated from conversion ofpyruvate to acetyl-CoA given that two molecules of pyruvate aregenerated per molecule of glucose metabolized via the EMP pathway. Thiscould be done by employing any of the following enzymes or enzyme setsto convert pyruvate to acetyl-CoA:

-   I. NAD-dependant pyruvate dehydrogenase;-   II. Pyruvate formate lyase and NAD-dependant formate dehydrogenase;-   III. Pyruvate:ferredoxin oxidoreductase and NADH:ferredoxin    oxidoreductase;-   IV. Pyruvate decarboxylase and an NAD-dependant acylating    acetylaldehyde dehydrogenase;-   V. Pyruvate decarboxylase, NAD-dependant acylating acetaldehyde    dehydrogenase, acetate kinase, and phosphotransacetylase; and-   VI. Pyruvate decarboxylase, NAD-dependant acylating acetaldehyde    dehydrogenase, and acetyl-CoA synthetase.

Overall, four molecules of NADH can be attained per glucose moleculemetabolized. In one aspect, the fatty alcohol pathway requires threereduction steps from acetyl-CoA. Therefore, it can be possible that eachof these three reduction steps will utilize NADPH or NADH as thereducing agents, in turn converting these molecules to NADP or NAD,respectively. Therefore, in some aspects, it can be desirable that allreduction steps are NADH-dependant in order to maximize the yield offatty alcohols, fatty aldehydes or fatty acis. High yields of fattyalcohols, fatty aldehydes and fatty acids can thus be accomplished by:

Identifying and implementing endogenous or exogenous fatty alcohol,fatty aldehyde or fatty acid pathway enzymes with a stronger preferencefor NADH than other reducing equivalents such as NADPH,

-   I. Attenuating one or more endogenous fatty alcohol, fatty aldehyde    or fatty acid pathway enzymes that contribute NADPH-dependant    reduction activity,-   II. Altering the cofactor specificity of endogenous or exogenous    fatty alcohol, fatty aldehyde or fatty acid pathway enzymes so that    they have a stronger preference for NADH than their natural    versions, or-   III. Altering the cofactor specificity of endogenous or exogenous    fatty alcohol, fatty aldehyde or fatty acid pathway enzymes so that    they have a weaker preference for NADPH than their natural versions.

The individual enzyme or protein activities from the endogenous orexogenous DNA sequences can be assayed using methods well known in theart. For example, the genes can be expressed in E. coli and the activityof their encoded proteins can be measured using cell extracts.Alternatively, the enzymes can be purified using standard procedureswell known in the art and assayed for activity. Spectrophotometric basedassays are particularly effective.

Several examples and methods of altering the cofactor specificity ofenzymes are known in the art. For example, Khoury et al. (Protein Sci.2009 October; 18(10): 2125-2138) created several xylose reductaseenzymes with an increased affinity for NADH and decreased affinity forNADPH. Ehsani et al (Biotechnology and Bioengineering, Volume 104, Issue2, pages 381-389, 1 Oct. 2009) drastically decreased activity of2,3-butanediol dehydrogenase on NADH while increasing activity on NADPH.Machielsen et al (Engineering in Life Sciences, Volume 9, Issue 1, pages38-44, February 2009) dramatically increased activity of alcoholdehydrogenase on NADH. Khoury et al (Protein Sci. 2009 October; 18(10):2125-2138) list in Table I several previous examples of successfullychanging the cofactor preference of over 25 other enzymes. Additionaldescriptions can be found in Lutz et al, Protein Engineering Handbook,Volume 1 and Volume 2, 2009, Wiley-VCH Verlag GmbH & Co. KGaA, inparticular, Chapter 31: Altering Enzyme Substrate and CofactorSpecificity via Protein Engineering.

Example VI Determining Cofactor Preference of Pathway Enzymes

This example describes an experimental method for determining thecofactor preference of an enzyme.

Cofactor preference of enzymes for each of the pathway steps can bedetermined by cloning the individual genes on a plasmid behind aconstitutive or inducible promoter and transforming into a host organismsuch as Escherichia coli. For example, genes encoding enzymes thatcatalyze pathway steps from: 1) acetoacetyl-CoA to 3-hydroxybutyryl-CoA,2) 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde, 3)3-hydroxybutyraldehyde to 1,3-butanediol (wherein R₁ is C₁; R₃ is OH)can be assembled onto the pZ-based expression vectors as describedbelow.

Replacement of the Stuffer Fragment in the pZ-Based Expression Vectors.

Vector backbones were obtained from Dr. Rolf Lutz of Expressys(http://www.expressys.de/). The vectors and strains are based on the pZExpression System developed by Lutz and Bujard (Nucleic Acids Res 25,1203-1210 (1997)). The pZE13luc, pZA33luc, pZS*13luc and pZE22luccontain the luciferase gene as a stuffer fragment. To replace theluciferase stuffer fragment with a lacZ-alpha fragment flanked byappropriate restriction enzyme sites, the luciferase stuffer fragment isremoved from each vector by digestion with EcoRI and XbaI. ThelacZ-alpha fragment is PCR amplified from pUC19 with the followingprimers:

lacZalpha-RI (SEQ ID NO: ) 5′GACGAATTCGCTAGCAAGAGGAGAAGTCGACATGTCCAATTCACTGGCC GTCGTTTTAC3′lacZalpha 3′BB (SEQ ID NO: ) 5′-GACCCTAGGAAGCTTTCTAGAGTCGACCTATGCGGCATCAGAGCAGA-3′

This generates a fragment with a 5′ end of EcoRI site, NheI site, aRibosomal Binding Site, a SalI site and the start codon. On the 3′ endof the fragment are the stop codon, XbaI, HindIII, and AvrII sites. ThePCR product is digested with EcoRI and AvrII and ligated into the basevectors digested with EcoRI and XbaI (XbaI and AvrII have compatibleends and generate a non-site). Because NheI and XbaI restriction enzymesites generate compatible ends that can be ligated together (butgenerate a site after ligation that is not digested by either enzyme),the genes cloned into the vectors can be “Biobricked” together(http://openwetware.org/wiki/Synthetic_Biology:BioBricks). Briefly, thismethod enables joining an unlimited number of genes into the vectorusing the same 2 restriction sites (as long as the sites do not appearinternal to the genes), because the sites between the genes aredestroyed after each addition. These vectors can be subsequentlymodified using the Phusion® Site-Directed Mutagenesis Kit (NEB, Ipswich,Mass., USA) to insert the spacer sequence AATTAA between the EcoRI andNheI sites. This eliminates a putative stem loop structure in the RNAthat bound the RBS and start codon.

All vectors have the pZ designation followed by letters and numbersindicating the origin of replication, antibiotic resistance marker andpromoter/regulatory unit. The origin of replication is the second letterand is denoted by E for ColE1, A for p15A and S for pSC101 (as well as alower copy number version of pSC101 designated S*)-based origins. Thefirst number represents the antibiotic resistance marker (1 forAmpicillin, 2 for Kanamycin, 3 for Chloramphenicol). The final numberdefines the promoter that regulated the gene of interest (1 forPLtetO-1, 2 for PLlacO-1 and 3 for PA1lacO-1). For the work discussedhere we employed three base vectors, pZS*13S, pZA33S and pZE13S,modified for the biobricks insertions as discussed above.

Plasmids containing genes encoding pathway enzymes can then transformedinto host strains containing lacIQ, which allow inducible expression byaddition of isopropyl β-D-1-thiogalactopyranoside (IPTG). Activities ofthe heterologous enzymes are tested in in vitro assays, using strain E.coli MG1655 lacIQ as the host for the plasmid constructs containing thepathway genes. Cells can be grown aerobically in LB media (Difco)containing the appropriate antibiotics for each construct, and inducedby addition of IPTG at 1 mM when the optical density (OD600) reachedapproximately 0.5. Cells can be harvested after 6 hours, and enzymeassays conducted as discussed below.

In Vitro Enzyme Assays.

To obtain crude extracts for activity assays, cells can be harvested bycentrifugation at 4,500 rpm (Beckman-Coulter, Allegera X-15R) for 10min. The pellets are resuspended in 0.3 mL BugBuster (Novagen) reagentwith benzonase and lysozyme, and lysis proceeds for about 15 minutes atroom temperature with gentle shaking Cell-free lysate is obtained bycentrifugation at 14,000 rpm (Eppendorf centrifuge 5402) for 30 min at4° C. Cell protein in the sample is determined using the method ofBradford et al., Anal. Biochem. 72:248-254 (1976), and specific enzymeassays conducted as described below. Activities are reported in Units/mgprotein, where a unit of activity is defined as the amount of enzymerequired to convert 1 micromol of substrate in 1 minute at roomtemperature.

Pathway steps can be assayed in the reductive direction using aprocedure adapted from several literature sources (Durre et al., FEMSMicrobiol. Rev. 17:251-262 (1995); Palosaari and Rogers, Bacteriol.170:2971-2976 (1988) and Welch et al., Arch. Biochem. Biophys.273:309-318 (1989). The oxidation of NADH or NADPH can be followed byreading absorbance at 340 nM every four seconds for a total of 240seconds at room temperature. The reductive assays can be performed in100 mM MOPS (adjusted to pH 7.5 with KOH), 0.4 mM NADH or 0.4 mM NADPH,and from 1 to 50 μmol of cell extract. For carboxylic acidreductase-like enzymes, ATP can also be added at saturatingconcentrations. The reaction can be started by adding the followingreagents: 100 μmol of 100 mM acetoacetyl-CoA, 3-hydroxybutyryl-CoA,3-hydroxybutyrate, or 3-hydroxybutyraldehyde. The spectrophotometer isquickly blanked and then the kinetic read is started. The resultingslope of the reduction in absorbance at 340 nM per minute, along withthe molar extinction coefficient of NAD(P)H at 340 nM (6000) and theprotein concentration of the extract, can be used to determine thespecific activity.

Example VII Methods for Increasing NADPH Availability

In some aspects of the invention, it can be advantageous to employpathway enzymes that have activity using NADPH as the reducing agent.For example, NADPH-dependant pathway enzymes can be highly specific forMI-FAE cycle, MD-FAE cycle and/or termination pathway intermediates orcan possess favorable kinetic properties using NADPH as a substrate. Ifone or more pathway steps is NADPH dependant, several alternativeapproaches to increase NADPH availability can be employed. Theseinclude:

-   -   1) Increasing flux relative to wild-type through the oxidative        branch of the pentose phosphate pathway comprising        glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase,        and 6-phosphogluconate dehydrogenase (decarboxylating). This        will generate 2 NADPH molecules per glucose-6-phosphate        metabolized. However, the decarboxylation step will reduce the        maximum theoretical yield of 1,3-butanediol.    -   2) Increasing flux relative to wild-type through the Entner        Doudoroff pathway comprising glucose-6-phosphate dehydrogenase,        6-phosphogluconolactonase, phosphogluconate dehydratase, and        2-keto-3-deoxygluconate 6-phosphate aldolase.    -   3) Introducing a soluble transhydrogenase to convert NADH to        NADPH.    -   4) Introducing a membrane-bound transhydrogenase to convert NADH        to NADPH.    -   5) Employing an NADP-dependant glyceraldehyde-3-phosphate        dehydrogenase.    -   6) Employing any of the following enzymes or enzyme sets to        convert pyruvate to acetyl-CoA        -   a) NADP-dependant pyruvate dehydrogenase;        -   b) Pyruvate formate lyase and NADP-dependant formate            dehydrogenase;        -   c) Pyruvate:ferredoxin oxidoreductase and NADPH:ferredoxin            oxidoreductase;        -   d) Pyruvate decarboxylase and an NADP-dependant acylating            acetylaldehyde dehydrogenase;        -   e) Pyruvate decarboxylase, NADP-dependant acetaldehyde            dehydrogenase, acetate kinase, and phosphotransacetylase;            and        -   f) Pyruvate decarboxylase, NADP-dependant acetaldehyde            dehydrogenase, and acetyl-CoA synthetase; and optionally            attenuating NAD-dependant versions of these enzymes.    -   7) Altering the cofactor specificity of a native        glyceraldehyde-3-phosphate dehydrogenase, pyruvate        dehydrogenase, formate dehydrogenase, or acylating        acetylaldehyde dehydrogenase to have a stronger preference for        NADPH than their natural versions.    -   8) Altering the cofactor specificity of a native        glyceraldehyde-3-phosphate dehydrogenase, pyruvate        dehydrogenase, formate dehydrogenase, or acylating        acetylaldehyde dehydrogenase to have a weaker preference for        NADH than their natural versions.

The individual enzyme or protein activities from the endogenous orexogenous DNA sequences can be assayed using methods well known in theart. For example, the genes can be expressed in E. coli and the activityof their encoded proteins can be measured using cell extracts asdescribed in the previous example. Alternatively, the enzymes can bepurified using standard procedures well known in the art and assayed foractivity. Spectrophotometric based assays are particularly effective.

Several examples and methods of altering the cofactor specificity ofenzymes are known in the art. For example, Khoury et al (Protein Sci.2009 October; 18(10): 2125-2138) created several xylose reductaseenzymes with an increased affinity for NADH and decreased affinity forNADPH. Ehsani et al (Biotechnology and Bioengineering, Volume 104, Issue2, pages 381-389, 1 Oct. 2009) drastically decreased activity of2,3-butanediol dehydrogenase on NADH while increasing activity on NADPH.Machielsen et al (Engineering in Life Sciences, Volume 9, Issue 1, pages38-44, February 2009) dramatically increased activity of alcoholdehydrogenase on NADH. Khoury et al (Protein Sci. 2009 October; 18(10):2125-2138) list in Table I several previous examples of successfullychanging the cofactor preference of over 25 other enzymes. Additionaldescriptions can be found in Lutz et al, Protein Engineering Handbook,Volume 1 and Volume 2, 2009, Wiley-VCH Verlag GmbH & Co. KGaA, inparticular, Chapter 31: Altering Enzyme Substrate and CofactorSpecificity via Protein Engineering.

Enzyme candidates for these steps are provided below.

Glucose-6-Phosphate Dehydrogenase

Protein GenBank ID GI Number Organism ZWF1 NP_014158.1 6324088Saccharomyces cerevisiae S288c ZWF1 XP_504275.1 50553728 Yarrowialipolytica Zwf XP_002548953.1 255728055 Candida tropicalis MYA-3404 ZwfXP_001400342.1 145233939 Aspergillus niger CBS 513.88 KLLA0D19855gXP_453944.1 50307901 Kluyveromyces lactis NRRL Y-1140

6-Phosphogluconolactonase

Protein GenBank ID GI Number Organism SOL3 NP_012033.2 82795254Saccharomyces cerevisiae S288c SOL4 NP_011764.1 6321687 Saccharomycescerevisiae S288c YALI0E11671g XP_503830.1 50552840 Yarrowia lipolyticaYALI0C19085g XP_501998.1 50549055 Yarrowia lipolytica ANI_1_656014XP_001388941.1 145229265 Aspergillus niger CBS 513.88 CTRG_00665XP_002545884.1 255721899 Candida tropicalis MYA-3404 CTRG_02095XP_002547788.1 255725718 Candida tropicalis MYA-3404 KLLA0A05390gXP_451238.1 50302605 Kluyveromyces lactis NRRL Y-1140 KLLA0C08415gXP_452574.1 50305231 Kluyveromyces lactis NRRL Y-1140

6-Phosphogluconate Dehydrogenase (Decarboxylating)

Protein GenBank ID GI Number Organism GND1 NP_012053.1 6321977Saccharomyces cerevisiae S288c GND2 NP_011772.1 6321695 Saccharomycescerevisiae S288c ANI_1_282094 XP_001394208.2 317032184 Aspergillus nigerCBS 513.88 ANI_1_2126094 XP_001394596.2 317032939 Aspergillus niger CBS513.88 YALI0B15598g XP_500938.1 50546937 Yarrowia lipolytica CTRG_03660XP_002549363.1 255728875 Candida tropicalis MYA-3404 KLLA0A09339gXP_451408.1 50302941 Kluyveromyces lactis NRRL Y-1140

Phosphogluconate Dehydratase

Protein GenBank ID GI Number Organism Edd AAC74921.1 1788157 Escherichiacoli K-12 MG1655 Edd AAG29866.1 11095426 Zymomonas mobilis subsp.mobilis ZM4 Edd YP_350103.1 77460596 Pseudomonas fluorescens Pf0-1ANI_1_2126094 XP_001394596.2 317032939 Aspergillus niger CBS 513.88YALI0B15598g XP_500938.1 50546937 Yarrowia lipolytica CTRG_03660XP_002549363.1 255728875 Candida tropicalis MYA-3404 KLLA0A09339gXP_451408.1 50302941 Kluyveromyces lactis NRRL Y-1140

2-Keto-3-Deoxygluconate 6-Phosphate Aldolase

Protein GenBank ID GI Number Organism Eda NP_416364.1 16129803Escherichia coli K-12 MG1655 Eda Q00384.2 59802878 Zymomonas mobilissubsp. mobilis ZM4 Eda ABA76098.1 77384585 Pseudomonas fluorescens Pf0-1

Soluble Transhydrogenase

Protein GenBank ID GI Number Organism SthA NP_418397.2 90111670Escherichia coli K-12 MG1655 SthA YP_002798658.1 226943585 Azotobactervinelandii DJ SthA O05139.3 11135075 Pseudomonas fluorescens

Membrane-Bound Transhydrogenase

Protein GenBank ID GI Number Organism ANI_1_29100 XP_001400109.2317027842 Aspergillus niger CBS 513.88 Pc21g18800 XP_002568871.1226943585 255956237 Penicillium chrysogenum Wisconsin 54-1255 SthAO05139.3 11135075 Pseudomonas fluorescens NCU01140 XP_961047.2 164426165Neurospora crassa OR74A

NADP-Dependant Glyceraldehyde-3-Phosphate Dehydrogenase

Protein GenBank ID GI Number Organism gapN AAA91091.1 642667Streptococcus mutans NP-GAPDH AEC07555.1 330252461 Arabidopsis thalianaGAPN AAM77679.2 82469904 Triticum aestivum gapN CAI56300.1 87298962Clostridium acetobutylicum NADP-GAPDH 2D2I_A 112490271 Synechococcuselongatus PCC 7942 NADP-GAPDH CAA62619.1 4741714 Synechococcus elongatusPCC 7942 GDP1 XP_455496.1 50310947 Kluyveromyces lactis NRRL Y-1140HP1346 NP_208138.1 15645959 Helicobacter pylori 26695

NAD-Dependant Glyceraldehyde-3-Phosphate Dehydrogenase

Protein GenBank ID GI Number Organism TDH1 NP_012483.1 6322409Saccharomyces cerevisiae s288c TDH2 NP_012542.1 6322468 Saccharomycescerevisiae s288c TDH3 NP_011708.1 632163 Saccharomyces cerevisiae s288cKLLA0A11858g XP_451516.1 50303157 Kluyveromyces lactis NRRL Y-1140KLLA0F20988g XP_456022.1 50311981 Kluyveromyces lactis NRRL Y-1140ANI_1_256144 XP_001397496.1 145251966 Aspergillus niger CBS 513.88YALI0C06369g XP_501515.1 50548091 Yarrowia lipolytica CTRG_05666XP_002551368.1 255732890 Candida tropicalis MYA-3404

Mutated LpdA from E. coli K-12 MG1655described in Biochemistry, 1993, 32 (11), pp 2737-2740: (SEQ ID NO: )     MSTEIKTQVVVLGAGPAGYSAAFRCADLGLETVIVERYNTLGGVCLNVGCIPSKALLHVAKVIEEAKALAEHGIVFGEPKTDIDKIRTWKEKVINQLTGGLAGMAKGRKVKVVNGLGKFTGANTLEVEGENGKTVINFDNAIIAAGSRPIQLPFIPHEDPRIWDSTDALELKEVPERLLVMGGGIIGLEMGTVYHALGSQIDVVVRKHQVIRAADKDIVKVFTKRISKKFNLMLETKVTAVEAKEDGIYVTMEGKKAPAEPQRYDAVLVAIGRVPNGKNLDAGKAGVEVDDRGFIRVDKQLRTNVPHIFAIGDIVGQPMLAHKGVHEGHVAAEVIAGKKHYFDPKVIPSIAYTEPEVAWVGLTEKEAKEKGISYETATFPWAASGRAIASDCADGMTKLIFDKESHRVIGGAIVGTNGGELLGEIGLAIEMGCDAEDIALTIHAHPTLHESVGLAAEVFEGSITDLPNPKAKKK Mutated LpdA from E. coli K-12 MG1655described in Biochemistry, 1993, 32 (11), pp 2737-2740: (SEQ ID NO: )     MSTEIKTQVVVLGAGPAGYSAAFRCADLGLETVIVERYNTLGGVCLNVGCIPSKALLHVAKVIEEAKALAEHGIVFGEPKTDIDKIRTWKEKVINQLTGGLAGMAKGRKVKVVNGLGKFTGANTLEVEGENGKTVINFDNAIIAAGSRPIQLPFIPHEDPRIWDSTDALELKEVPERLLVMGGGIIALEMATVYHALGSQIDVVVRKHQVIRAADKDIVKVFTKRISKKFNLMLETKVTAVEAKEDGIYVTMEGKKAPAEPQRYDAVLVAIGRVPNGKNLDAGKAGVEVDDRGFIRVDKQLRTNVPHIFAIGDIVGQPMLAHKGVHEGHVAAEVIAGKKHYFDPKVIPSIAYTEPEVAWVGLTEKEAKEKGISYETATFPWAASGRAIASDCADGMTKLIFDKESHRVIGGAIVGTNGGELLGEIGLAIEMGCDAEDIALTIHAHPTLHESVGLAAEVFEGSITDLPNPKAKKK

NADP-Dependant Formate Dehydrogenase

Protein GenBank ID GI Number Organism fdh ACF35003. 194220249Burkholderia stabilis fdh ABC20599.2 146386149 Moorella thermoaceticaATCC 39073

Mutant Candida bodinii enzyme describedin Journal of Molecular Catalysis B: Enzymatic, Volume 61, Issues 3-4,December 2009, Pages 157-161: (SEQ ID NO: )     MKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGETSELDKHIPDADIIITTPFHPAYITKERLDKAKNLKLVVVAGVGSDHIDLDYINQTGKKISVLEVTGSNVVSVAEHVVMTMLVLVRNFVPAHEQIINHDWEVAAIAKDAYDIEGKTIATIGAGRIGYRVLERLLPFNPKELLYYQRQALPKEAEEKVGARRVENIEELVAQADIVTVNAPLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDVWFPQPAPKDHPWRDMRNKYGAGNAMTPHYSGTTLDAQTRYAEGTKNILESFFTGKFDYRPQD IILLNGEYVTKAYGKHDKKMutant Candida bodinii enzyme describedin Journal of Molecular Catalysis B: Enzymatic, Volume 61, Issues 3-4,December 2009, Pages 157-161: (SEQ ID NO: )     MKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGETSELDKHIPDADIIITTPFHPAYITKERLDKAKNLKLVVVAGVGSDHIDLDYINQTGKKISVLEVTGSNVVSVAEHVVMTMLVLVRNFVPAHEQIINHDWEVAAIAKDAYDIEGKTIATIGAGRIGYRVLERLLPFNPKELLYYSPQALPKEAEEKVGARRVENIEELVAQADIVTVNAPLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDVWFPQPAPKDHPWRDMRNKYGAGNAMTPHYSGTTLDAQTRYAEGTKNILESFFTGKFDYRPQD IILLNGEYVTKAYGKHDKKMutant Saccharomyces cerevisiae enzymedescribed in Biochem J. 2002 November 1:367(Pt. 3):841-847:(SEQ ID NO: )      MSKGKVLLVLYEGGKHAEEQEKLLGCIENELGIRNFIEEQGYELVTTIDKDPEPTSTVDRELKDAEIVITTPFFPAYISRNRIAEAPNLKLCVTAGVGSDHVDLEAANERKITVTEVTGSNVVSVAEHVMATILVLIRNYNGGHQQAINGEWDIAGVAKNEYDLEDKIISTVGAGRIGYRVLERLVAFNPKKLLYYARQELPAEAINRLNEASKLFNGRGDIVQRVEKLEDMVAQSDVVTINCPLHKDSRGLFNKKLISHMKDGAYLVNTARGAICVAEDVAEAVKSGKLAGYGGDVWDKQPAPKDHPWRTMDNKDHVGNAMTVHISGTSLDAQKRYAQGVKNILNSYFSKKFDYRPQDIIVQNGSYATRAYGQKK.

NADPH:Ferredoxin Oxidoreductase

Protein GenBank ID GI Number Organism petH YP_171276.1 56750575Synechococcus elongatus PCC 6301 fpr NP_457968.1 16762351 Salmonellaenterica fnr1 XP_001697352.1 159478523 Chlamydomonas reinhardtii rfnr1NP_567293.1 18412939 Arabidopsis thaliana aceF NP_414657.1 6128108Escherichia coli K-12 MG1655

NADP-Dependant Acylating Acetylaldehyde Dehydrogenase

Protein GenBank ID GI Number Organism adhB AAB06720.1 1513071Thermoanaerobacter pseudethanolicus ATCC 33223 TheetDRAFT_0840ZP_08211603. 326390041 Thermoanaerobacter ethanolicus JW 200 Cbei_3832YP_001310903.1 150018649 Clostridium beijerinckii NCIMB 8052 Cbei_4054YP_001311120.1 150018866 Clostridium beijerinckii NCIMB 8052 Cbei_4045YP_001311111.1 150018857 Clostridium beijerinckii NCIMB 8052

Exemplary genes encoding pyruvate dehydrogenase, pyruvate:ferredoxinoxidoreductase, pyruvate formate lyase, pyruvate decarboxylase, acetatekinase, phosphotransacetylase and acetyl-CoA synthetase are describedabove in Example II.

Example VIII Engineering Saccharomyces cerevisiae for ChemicalProduction

Eukaryotic hosts have several advantages over prokaryotic systems. Theyare able to support post-translational modifications and hostmembrane-anchored and organelle-specific enzymes. Genes in eukaryotestypically have introns, which can impact the timing of gene expressionand protein structure.

An exemplary eukaryotic organism well suited for industrial chemicalproduction is Saccharomyces cerevisiae. This organism is wellcharacterized, genetically tractable and industrially robust. Genes canbe readily inserted, deleted, replaced, overexpressed or underexpressedusing methods known in the art. Some methods are plasmid-based whereasothers modify the chromosome (Guthrie and Fink. Guide to Yeast Geneticsand Molecular and Cell Biology, Part B, Volume 350, Academic Press(2002); Guthrie and Fink, Guide to Yeast Genetics and Molecular and CellBiology, Part C, Volume 351, Academic Press (2002)).

Plasmid-mediated gene expression is enabled by yeast episomal plasmids(YEps). YEps allow for high levels of expression; however they are notvery stable and they require cultivation in selective media. They alsohave a high maintenance cost to the host metabolism. High copy numberplasmids using auxotrophic (e.g., URA3, TRP1, HIS3, LEU2) or antibioticselectable markers (e.g., Zeo^(R) or Kan^(R)) can be used, often withstrong, constitutive promoters such as PGK1 or ACT1 and a transcriptionterminator-polyadenylation region such as those from CYC1 or AOX. Manyexamples are available for one well-versed in the art. These includepVV214 (a 2 micron plasmid with URA3 selectable marker) and pVV200 (2micron plasmid with TRP1 selectable marker) (Van et al., Yeast20:739-746 (2003)). Alternatively, low copy plasmids such as centromericor CEN plamids can be used. Again, many examples are available for onewell-versed in the art. These include pRS313 and pRS315 (Sikorski andHieter, Genetics 122:19-27 (1989) both of which require that a promoter(e.g., PGK1 or ACT1) and a terminator (e.g., CYC1, AOX) are added.

For industrial applications, chromosomal overexpression of genes ispreferable to plasmid-mediated overexpression. Mikkelsen and coworkershave identified 11 integration sites on highly expressed regions of theS. cerevisiae genome on chromosomes X, XI and XII (Mikkelsen et al, MetEng 14:104-11 (2012)). The sites are separated by essential genes,minimizing the possibility of recombination between sites.

Tools for inserting genes into eukaryotic organisms such as S.cerevisiae are known in the art. Particularly useful tools include yeastintegrative plasmids (YIps), yeast artificial chromosomes (YACS) andgene targeting/homologous recombination. Note that these tools can alsobe used to insert, delete, replace, underexpress or otherwise alter thegenome of the host.

Yeast integrative plasmids (YIps) utilize the native yeast homologousrecombination system to efficiently integrate DNA into the chromosome.These plasmids do not contain an origin of replication and can thereforeonly be maintained after chromosomal integration. An exemplary constructincludes a promoter, the gene of interest, a terminator, and aselectable marker with a promoter, flanked by FRT sites, loxP sites, ordirect repeats enabling the removal and recycling of the resistancemarker. The method entails the synthesis and amplification of the geneof interest with suitable primers, followed by the digestion of the geneat a unique restriction site, such as that created by the EcoRI and XhoIenzymes (Vellanki et al., Biotechnol Lett. 29:313-318 (2007)). The geneof interest is inserted at the EcoRI and XhoI sites into a suitableexpression vector, downstream of the promoter. The gene insertion isverified by PCR and DNA sequence analysis. The recombinant plasmid isthen linearized and integrated at a desired site into the chromosomalDNA of S. cerevisiae using an appropriate transformation method. Thecells are plated on the YPD medium with an appropriate selection markerand incubated for 2-3 days. The transformants are analyzed for therequisite gene insert by colony PCR. To remove the antibiotic markerfrom a construct flanked by loxP sites, a plasmid containing the Crerecombinase is introduced. Cre recombinase promotes the excision ofsequences flanked by loxP sites. (Gueldener et al., Nucleic Acids Res30:e23 (2002)). The resulting strain is cured of the Cre plasmid bysuccessive culturing on media without any antibiotic present.Alternately, the Cre recombinase plasmid has a URA selection marker andthe plasmid is efficiently removed by growing cells on 5-FOA which actsas a counter-selection for URA. This method can also be employed for ascarless integration instead of using loxP. One skilled in the art canintegrate using URA as a marker, select for integration by growing onURA-minus plates, and then select for URA mutants by growing on 5-FOAplates. 5-FOA is converted to the toxic 5-fluoruracil by the URA geneproduct. Alternatively, the FLP-FRT system can be used to integrategenes into the chromosome. This system involves the recombination ofsequences between short Flipase Recognition Target (FRT) sites by theFlipase recombination enzyme (FLP) derived from the 2μ plasmid of theyeast Saccharomyces cerevisiae (Sadowski, P. D., Prog. Nucleic. Acid.Res. Mol. Biol. 51:53-91 (1995); Zhu and Sadowski J. Biol. Chem.270:23044-23054 (1995)). Similarly, gene deletion methodologies will becarried out as described in refs. Baudin et al. Nucleic. Acids Res.21:3329-3330 (1993); Brachmann et al., Yeast 14:115-132 (1998); Giaeveret al., Nature 418:387-391 (2002); Longtine et al., Yeast 14:953-961(1998) Winzeler et al., Science 285:901-906 (1999).

Another approach for manipulating the yeast chromosome is genetargeting. This approach takes advantage of the fact that doublestranded DNA breaks in yeast are repaired by homologous recombination.Linear DNA fragments flanked by targeting sequences can thus beefficiently integrated into the yeast genome using the native homologousrecombination machinery. In addition to the application of insertinggenes, gene targeting approaches are useful for genomic DNAmanipulations such as deleting genes, introducing mutations in a gene,its promoter or other regulatory elements, or adding a tag to a gene.

Yeast artificial chromosomes (YACs) are artificial chromosomes usefulfor pathway construction and assembly. YACs enable the expression oflarge sequences of DNA (100-3000 kB) containing multiple genes. The useof YACs was recently applied to engineer flavenoid biosynthesis in yeast(Naesby et al, Microb Cell Fact 8:49-56 (2009)). In this approach, YACswere used to rapidly test randomly assembled pathway genes to find thebest combination.

The expression level of a gene can be modulated by altering the sequenceof a gene and/or its regulatory regions. Such gene regulatory regionsinclude, for example, promoters, enhancers, introns, and terminators.Functional disruption of negative regulatory elements such as repressorsand/or silencers also can be employed to enhance gene expression. RNAbased tools can also be employed to regulate gene expression. Such toolsinclude RNA aptamers, riboswitches, antisense RNA, ribozymes andriboswitches.

For altering a gene's expression by its promoter, libraries ofconstitutive and inducible promoters of varying strengths are available.Strong constitutive promoters include pTEF1, pADH1 and promoters derivedfrom glycolytic pathway genes. The pGAL promoters are well-studiedinducible promoters activated by galactose and repressed by glucose.Another commonly used inducible promoter is the copper induciblepromoter pCUP1 (Farhi et al, Met Eng 13:474-81 (2011)). Furthervariation of promoter strengths can be introduced by mutagenesis orshuffling methods. For example, error prone PCR can be applied togenerate synthetic promoter libraries as shown by Alper and colleagues(Alper et al, PNAS 102:12678-83 (2005)). Promoter strength can becharacterized by reporter proteins such as beta-galactosidase,fluorescent proteins and luciferase.

The placement of an inserted gene in the genome can alter its expressionlevel. For example, overexpression of an integrated gene can be achievedby integrating the gene into repeating DNA elements such as ribosomalDNA or long terminal repeats.

For exogenous expression in yeast or other eukaryotic cells, genes canbe expressed in the cytosol without the addition of leader sequence, orcan be targeted to mitochondrion or other organelles, or targeted forsecretion, by the addition of a suitable targeting sequence such as amitochondrial targeting or secretion signal suitable for the host cells.Thus, it is understood that appropriate modifications to a nucleic acidsequence to remove or include a targeting sequence can be incorporatedinto an exogenous nucleic acid sequence to impart desirable properties.Genetic modifications can also be made to enhance polypeptide synthesis.For example, translation efficiency is enhanced by substituting ribosomebinding sites with an optimal or consensus sequence and/or altering thesequence of a gene to add or remove secondary structures. The rate oftranslation can also be increased by substituting one coding sequencewith another to better match the codon preference of the host.

Example IX Termination Pathways for Making Fatty Alcohols, Aldehydes andAcids

This example describes enzymes for converting intermediates of theMI-FAE cycle or MD-FAE cycle to products of interest such as fattyalcohols, fatty aldehydes, and fatty acids. Pathways are shown in FIGS.1 and 7. Enzymes for catalyzing steps A-G are disclosed in Example I.This example describes enzymes suitable for catalyzing steps H-N.

Enzymes include: A. Thiolase, B. 3-Ketoacyl-CoA reductase, C.β-Hydroxyl-ACP dehydratase, D. Enoyl-CoA reductase, E. Acyl-CoAreductase (aldehyde forming), F. Alcohol dehydrogenase, G. Acyl-CoAreductase (alcohol forming), H. acyl-CoA hydrolase, transferase orsynthetase, J. Acyl-ACP reductase, K. Acyl-CoA:ACP acyltransferase, L.Thioesterase, N. Aldehyde dehydrogenase (acid forming) or carboxylicacid reductase.

Pathways for converting an MI-FAE cycle intermediate to an fattyalcohol, fatty aldehyde or fatty acid product are shown in the tablebelow. These pathways are also referred to herein as “terminationpathways”.

Termination pathway Product enzymes from FIG. 1 Acid H K/L E/N K/J/NAldehyde E K/J H/N K/L/N Alcohol E/F K/J/F H/N/F K/L/N/F G

Product specificity can be fine-tuned using one or more enzymes shown inFIGS. 1 and 6. Chain length is controlled by one or more enzymes of theelongation pathway in conjunction with one more enzymes of thetermination pathway as described above. The structure of the product iscontrolled by one or more enzymes of the termination pathway.

Examples of selected termination pathway enzymes reacting with variouspathway intermediates are shown in the table below. Additional examplesare described herein.

Enzyme Substrate Example Acyl-CoA reductase Acyl-CoA Acr1 of A. bayliyi(GenBank AAC45217) 3-Hydroxyacyl- PduP of L. reuteri CoA (GenBankCCC03595.1) 3-Oxoacyl-CoA Mcr of S. tokodaii (GenBank NP_378167)Acyl-CoA hydrolase, Acyl-CoA tesB of E. coli transferase or synthetase(GenBank NP_414986) 3-Hydroxyacyl- hibch of R. norvegicus CoA (GenBankQ5XIE6.2) 3-Oxoacyl-CoA MKS2 of S. lycopersicum (GenBank ACG69783)Enoyl-CoA gctAB of Acidaminococcus fermentans (GenBank CAA57199,CAA57200) Acyl-ACP Acyl-CoA fabH of E. coli acyltransferase (GenBankAAC74175.1)

Step H. Acyl-CoA Hydrolase, Transferase or Synthase

Acyl-CoA hydrolase, transferase and synthase enzymes convert acyl-CoAmoieties to their corresponding acids. Such an enzyme can be utilized toconvert, for example, a fatty acyl-CoA to a fatty acid, a3-hydroxyacyl-CoA to a 3-hydroxyacid, a 3-oxoacyl-CoA to a 3-oxoacid, oran enoyl-CoA to an enoic acid.

CoA hydrolase or thioesterase enzymes in the 3.1.2 family hydrolyzeacyl-CoA molecules to their corresponding acids. Several CoA hydrolaseswith different substrate ranges are suitable for hydrolyzing acyl-CoA,3-hydroxyacyl-CoA, 3-oxoacyl-CoA and enoyl-CoA substrates to theircorresponding acids. For example, the enzyme encoded by acot12 fromRattus norvegicus brain (Robinson et al., Biochem. Biophys. Res. Commun.71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA andmalonyl-CoA. The human dicarboxylic acid thioesterase, encoded by acot8,exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA,and dodecanedioyl-CoA (Westin et al., J. Biol. Chem. 280:38125-38132(2005)). The closest E. coli homolog to this enzyme, tesB, can alsohydrolyze a range of CoA thiolesters (Naggert et al., J Biol Chem266:11044-11050 (1991)). A similar enzyme has also been characterized inthe rat liver (Deana R., Biochem Int 26:767-773 (1992)). Additionalenzymes with hydrolase activity in E. coli include ybgC, paaI, and ybdB(Kuznetsova, et al., FEMS Microbiol Rev, 2005, 29(2):263-279; Song etal., J Biol Chem, 2006, 281(16):11028-38). Though its sequence has notbeen reported, the enzyme from the mitochondrion of the pea leaf has abroad substrate specificity, with demonstrated activity on acetyl-CoA,propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, andcrotonyl-CoA (Zeiher et al., Plant. Physiol. 94:20-27 (1990)) Theacetyl-CoA hydrolase, ACH1, from S. cerevisiae represents anothercandidate hydrolase (Buu et al., J. Biol. Chem. 278:17203-17209 (2003)).Additional enzymes with aryl-CoA hydrolase activity include thepalmitoyl-CoA hydrolase of Mycobacterium tuberculosis (Wang et al.,Chem. Biol. 14:543-551 (2007)) and the acyl-CoA hydrolase of E. coliencoded by entH (Guo et al., Biochemistry 48:1712-1722 (2009)).Additional CoA hydrolase enzymes are described above.

GenBank Gene name Accession # GI# Organism acot12 NP_570103.1 18543355Rattus norvegicus tesB NP_414986 16128437 Escherichia coli acot8CAA15502 3191970 Homo sapiens acot8 NP_570112 51036669 Rattus norvegicustesA NP_415027 16128478 Escherichia coli ybgC NP_415264 16128711Escherichia coli paaI NP_415914 16129357 Escherichia coli ybdB NP_41512916128580 Escherichia coli ACH1 NP_009538 6319456 Saccharomycescerevisiae Rv0098 NP_214612.1 15607240 Mycobacterium tuberculosis entHAAC73698.1 1786813 Escherichia coli

CoA hydrolase enzymes active on 3-hydroxyacyl-CoA, 3-oxoacyl-CoA andenoyl-CoA intermediates are also well known in the art. For example, anenzyme for converting enoyl-CoA substrates to their corresponding acidsis the glutaconate CoA-transferase from Acidaminococcus fermentans. Thisenzyme was transformed by site-directed mutagenesis into an acyl-CoAhydrolase with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA(Mack et al., FEBS. Lett. 405:209-212 (1997)). Another suitable enzymeis the fadM thioesterase III of E. coli. This enzyme is involved inoleate beta-oxidation and the preferred substrate is3,5-tetradecadienoyl-CoA (Nie et al, Biochem 47:7744-51 (2008)).

Protein GenBank ID GI Number Organism gctA CAA57199.1 559392Acidaminococcus fermentans gctB CAA57200.1 559393 Acidaminococcusfermentans gctA ACJ24333.1 212292816 Clostridium symbiosum gctBACJ24326.1 212292808 Clostridium symbiosum gctA NP_603109.1 19703547Fusobacterium nucleatum gctB NP_603110.1 19703548 Fusobacteriumnucleatum fadM NP_414977.1 16128428 Escherichia coli

3-Hydroxyisobutyryl-CoA hydrolase is active on 3-hydroxyacyl-CoAsubstrates (Shimomura et al., J Biol Chem. 269:14248-14253 (1994)).Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomuraet al., Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomuraet al., supra). Similar gene candidates can also be identified bysequence homology, including hibch of Saccharomyces cerevisiae and BC2292 of Bacillus cereus. An exemplary 3-oxoacyl-CoA hydrolase is MKS2 ofSolanum lycopersicum (Yu et al, Plant Physiol 154:67-77 (2010)). Thenative substrate of this enzyme is 3-oxo-myristoyl-CoA, which produces aC14 chain length product.

GenBank Gene name Accession # GI# Organism fadM NP_414977.1 16128428Escherichia coli hibch Q5XIE6.2 146324906 Rattus norvegicus hibchQ6NVY1.2 146324905 Homo sapiens hibch P28817.2 2506374 Saccharomycescerevisiae BC_2292 AP09256 29895975 Bacillus cereus MKS2 ACG69783.1196122243 Solanum lycopersicum

CoA transferases catalyze the reversible transfer of a CoA moiety fromone molecule to another. Several transformations require a CoAtransferase to activate carboxylic acids to their corresponding acyl-CoAderivatives. CoA transferase enzymes have been described in the openliterature and represent suitable candidates for these steps. These aredescribed below.

The gene products of cat1, cat2, and cat3 of Clostridium kluyveri havebeen shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, andbutyryl-CoA transferase activity, respectively (Seedorf et al., Proc.Natl. Acad. Sci U.S.A 105:2128-2133 (2008); Sohling et al., J Bacteriol.178:871-880 (1996)). Similar CoA transferase activities are also presentin Trichomonas vaginalis, Trypanosoma brucei, Clostridium aminobutyricumand Porphyromonas gingivalis (Riviere et al., J. Biol. Chem.279:45337-45346 (2004); van Grinsven et al., J. Biol. Chem.283:1411-1418 (2008)).

Protein GenBank ID GI Number Organism cat1 P38946.1 729048 Clostridiumkluyveri cat2 P38942.2 172046066 Clostridium kluyveri cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei cat2 CAB60036.1 6249316 Clostridium aminobutyricum cat2NP_906037.1 34541558 Porphyromonas gingivalis W83

A fatty acyl-CoA transferase that utilizes acetyl-CoA as the CoA donoris acetoacetyl-CoA transferase, encoded by the E. coli atoA (alphasubunit) and atoD (beta subunit) genes (Korolev et al., ActaCrystallogr. D. Biol. Crystallogr. 58:2116-2121 (2002); Vanderwinkel etal., 33:902-908 (1968)). This enzyme has a broad substrate range onsubstrates of chain length C3-C6 (Sramek et al., Arch Biochem Biophys171:14-26 (1975)) and has been shown to transfer the CoA moiety toacetate from a variety of branched and linear 3-oxo and acyl-CoAsubstrates, including isobutyrate (Matthies et al., Appl Environ.Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al., Biochem.Biophys. Res. Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel etal., Biochem. Biophys. Res. Commun. 33:902-908 (1968)). This enzyme isinduced at the transcriptional level by acetoacetate, so modification ofregulatory control may be necessary for engineering this enzyme into apathway (Pauli et al., Eur. J Biochem. 29:553-562 (1972)). Similarenzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al.,68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., ApplEnviron Microbiol 56:1576-1583 (1990); Wiesenborn et al., Appl EnvironMicrobiol 55:323-329 (1989)), and Clostridium saccharoperbutylacetonicum(Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)).

Gene GI # Accession No. Organism atoA 2492994 P76459.1 Escherichia coliatoD 2492990 P76458.1 Escherichia coli actA 62391407 YP_226809.1Corynebacterium glutamicum cg0592 62389399 YP_224801.1 Corynebacteriumglutamicum ctfA 15004866 NP_149326.1 Clostridium acetobutylicum ctfB15004867 NP_149327.1 Clostridium acetobutylicum ctfA 31075384 AAP42564.1Clostridium saccharoperbutylacetonicum ctfB 31075385 AAP42565.1Clostridium saccharoperbutylacetonicum

Beta-ketoadipyl-CoA transferase, also known assuccinyl-CoA:3:oxoacid-CoA transferase, is active on 3-oxoacyl-CoAsubstrates. This enzyme is encoded by pcaI and pcaJ Pseudomonas putida(Kaschabek et al., J Bacteriol. 184:207-215 (2002)). Similar enzymes arefound in Acinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30(1994)), Streptomyces coelicolor and Pseudomonas knackmussii (formerlysp. B13) (Gobel et al., J Bacteriol. 184:216-223 (2002); Kaschabek etal., J Bacteriol. 184:207-215 (2002)). Additional exemplarysuccinyl-CoA:3:oxoacid-CoA transferases have been characterized in inHelicobacter pylori (Corthesy-Theulaz et al., J Biol. Chem.272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein Expr.Purif. 53:396-403 (2007)) and Homo sapiens (Fukao, T., et al., Genomics68:144-151 (2000); Tanaka, H., et al., Mol Hum Reprod 8:16-23 (2002)).Genbank information related to these genes is summarized below.

Gene GI # Accession No. Organism pcaI 24985644 AAN69545.1 Pseudomonasputida pcaJ 26990657 NP_746082.1 Pseudomonas putida pcaI 50084858YP_046368.1 Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1 Acinetobactersp. ADP1 pcaI 21224997 NP_630776.1 Streptomyces coelicolor pcaJ 21224996NP_630775.1 Streptomyces coelicolor catI 75404583 Q8VPF3 Pseudomonasknackmussii catJ 75404582 Q8VPF2 Pseudomonas knackmussii HPAG1_0676108563101 YP_627417 Helicobacter pylori HPAG1_0677 108563102 YP_627418Helicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis ScoB16080949 NP_391777 Bacillus subtilis OXCT1 NP_000427 4557817 Homosapiens OXCT2 NP_071403 11545841 Homo sapiens

The conversion of acyl-CoA substrates to their acid products can becatalyzed by a CoA acid-thiol ligase or CoA synthetase in the 6.2.1family of enzymes. CoA synthases that convert ATP to ADP (ADP-forming)are reversible and react in the direction of acid formation, whereas AMPforming enzymes only catalyze the activation of an acid to an acyl-CoA.For fatty acid formation, deletion or attenuation of AMP forming enzymeswill reduce backflux. ADP-forming acetyl-CoA synthetase (ACD, EC6.2.1.13) is an enzyme that couples the conversion of acyl-CoA esters totheir corresponding acids with the concomitant synthesis of ATP. ACD Ifrom Archaeoglobus fulgidus, encoded by AF1211, was shown to operate ona variety of linear and branched-chain substrates including isobutyrate,isopentanoate, and fumarate (Musfeldt et al., J Bacteriol. 184:636-644(2002)). A second reversible ACD in Archaeoglobus fulgidus, encoded byAF1983, was also shown to have a broad substrate range (Musfeldt andSchonheit, J Bacteriol. 184:636-644 (2002)). The enzyme from Haloarculamarismortui (annotated as a succinyl-CoA synthetase) accepts propionate,butyrate, and branched-chain acids (isovalerate and isobutyrate) assubstrates, and was shown to operate in the forward and reversedirections (Brasen et al., Arch Microbiol 182:277-287 (2004)). The ACDencoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculumaerophilum showed the broadest substrate range of all characterizedACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) andphenylacetyl-CoA (Brasen et al, supra). Directed evolution orengineering can be used to modify this enzyme to operate at thephysiological temperature of the host organism. The enzymes from A.fulgidus, H. marismortui and P. aerophilum have all been cloned,functionally expressed, and characterized in E. coli (Brasen andSchonheit, supra; Musfeldt and Schonheit, J Bacteriol. 184:636-644(2002)). An additional candidate is succinyl-CoA synthetase, encoded bysucCD of E. coli and LSC1 and LSC2 genes of Saccharomyces cerevisiae.These enzymes catalyze the formation of succinyl-CoA from succinate withthe concomitant consumption of one ATP in a reaction which is reversiblein vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). The acyl CoAligase from Pseudomonas putida has been demonstrated to work on severalaliphatic substrates including acetic, propionic, butyric, valeric,hexanoic, heptanoic, and octanoic acids and on aromatic compounds suchas phenylacetic and phenoxyacetic acids (Fernandez-Valverde et al.,Appl. Environ. Microbiol. 59:1149-1154 (1993)). A related enzyme,malonyl CoA synthetase (6.3.4.9) from Rhizobium leguminosarum couldconvert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-,dimethyl-, cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, andbenzyl-malonate into their corresponding monothioesters (Pohl et al., J.Am. Chem. Soc. 123:5822-5823 (2001)).

Protein GenBank ID GI Number Organism AF1211 NP_070039.1 11498810Archaeoglobus fulgidus AF1983 NP_070807.1 11499565 Archaeoglobusfulgidus scs YP_135572.1 55377722 Haloarcula marismortui PAE3250NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2 sucC NP_415256.116128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia coli LSC1NP_014785 6324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683Saccharomyces cerevisiae paaF AAC24333.2 22711873 Pseudomonas putidamatB AAC83455.1 3982573 Rhizobium leguminosarum

Step J. Acyl-ACP Reductase

The reduction of an acyl-ACP to its corresponding aldehyde is catalyzedby an acyl-ACP reductase (AAR). Such a transformation is depicted instep J of FIGS. 1 and 7. Suitable enzyme candidates include the orf1594gene product of Synechococcus elongatus PCC7942 and homologs thereof(Schirmer et al, Science, 329: 559-62 (2010)). The S. elongates PCC7942acyl-ACP reductase is coexpressed with an aldehyde decarbonylase in anoperon that appears to be conserved in a majority of cyanobacterialorganisms. This enzyme, expressed in E. coli together with the aldehydedecarbonylase, conferred the ability to produce alkanes. The P. marinusAAR was also cloned into E. coli and, together with a decarbonylase,demonstrated to produce alkanes (US Application 2011/0207203).

Protein GenBank ID GI Number Organism orf1594 YP_400611.1 81300403Synechococcus elongatus PCC7942 PMT9312_0533 YP_397030.1 78778918Prochlorococcus marinus MIT 9312 syc0051_d YP_170761.1 56750060Synechococcus elongatus PCC 6301 Ava_2534 YP_323044.1 75908748 Anabaenavariabilis ATCC 29413 alr5284 NP_489324.1 17232776 Nostoc sp. PCC 7120Aazo_3370 YP_003722151.1 298491974 Nostoc azollae Cyan7425_0399YP_002481152.1 220905841 Cyanothece sp. PCC 7425 N9414_21225ZP_01628095.1 119508943 Nodularia spumigena CCY9414 L8106_07064ZP_01619574.1 119485189 Lyngbya sp. PCC 8106

Step K. Acyl-CoA:ACP Acyltransferase

The transfer of an acyl-CoA to an acyl-ACP is catalyzed byacyltransferase enzymes in EC class 2.3.1. Enzymes with this activityare described above.

Step L. Thioesterase

Acyl-ACP thioesterase enzymes convert an acyl-ACP to its correspondingacid. Such a transformation is required in step L of FIG. 1. Exemplaryenzymes include the FatA and FatB isoforms of Arabidopsis thaliana(Salas et al, Arch Biochem Biophys 403:25-34 (2002)). The activities ofthese two proteins vary with carbon chain length, with FatA preferringoleyl-ACP and FatB preferring palmitoyl-ACP. A number of thioesteraseswith different chain length specificities are listed in WO 2008/113041and are included in the table below. For example, it has been shownpreviously that expression of medium chain plant thioesterases like FatBfrom Umbellularia californica in E. coli results in accumulation of highlevels of medium chain fatty acids, primarily laurate (C12:0).Similarly, expression of Cuphea palustris FatB1 thioesterase in E. coliled to accumulation of C8-10:0 products (Dehesh et al, Plant Physiol110:203-10 (1996)). Similarly, Carthamus tinctorius thioesteraseexpressed in E. coli leads to >50 fold elevation in C 18:1 chaintermination and release as free fatty acid (Knutzon et al, Plant Physiol100:1751-58 (1992)). Methods for altering the substrate specificity ofthioesterases are also known in the art (for example, EP1605048).

Protein GenBank ID GI Number Organism fatA AEE76980.1 332643459Arabidopsis thaliana fatB AEE28300.1 332190179 Arabidopsis thalianafatB2 AAC49269.1 1292906 Cuphea hookeriana fatB3 AAC72881.1 3859828Cuphea hookeriana fatB1 AAC49179.1 1215718 Cuphea palustris M96568.1:AAA33019.1 404026 Carthamus tinctorius 94 . . . 1251 fatB1 Q41635.18469218 Umbellularia californica tesA AAC73596.1 1786702 Escherichiacoli

Step N. Aldehyde Dehydrogenase (Acid Forming) or Carboxylic AcidReductase

The conversion of an aldehyde to an acid is catalyzed by an acid-formingaldehyde dehydrogenase. Several Saccharomyces cerevisiae enzymescatalyze the oxidation of aldehydes to acids including ALD1 (ALD6), ALD2and ALD3 (Navarro-Avino et al, Yeast 15:829-42 (1999); Quash et al,Biochem Pharmacol 64:1279-92 (2002)). The mitochondrial proteins ALD4and ALD5 catalyze similar transformations (Wang et al, J Bacteriol180:822-30 (1998); Boubekeur et al, Eur J Biochem 268:5057-65 (2001)).HFD1 encodes a hexadecanal dehydrogenase. Exemplary acid-formingaldehyde dehydrogenase enzymes are listed in the table below.

Protein GenBank ID GI number Organism ALD2 NP_013893.1 6323822Saccharomyces cerevisiae s288c ALD3 NP_013892.1 6323821 Saccharomycescerevisiae s288c ALD4 NP_015019.1 6324950 Saccharomyces cerevisiae s288cALD5 NP_010996.2 330443526 Saccharomyces cerevisiae s288c ALD6NP_015264.1 6325196 Saccharomyces cerevisiae s288c HFD1 NP_013828.16323757 Saccharomyces cerevisiae s288c CaO19.8361 XP_710976.1 68490403Candida albicans CaO19.742 XP_710989.1 68490378 Candida albicansYALI0C03025 CAG81682.1 49647250 Yarrowia lipolytica ANI_1_1334164XP_001398871.1 145255133 Aspergillus niger ANI_1_2234074 XP_001392964.2317031176 Aspergillus niger ANI_1_226174 XP_001402476.1 145256256Aspergillus niger ALDH P41751.1 1169291 Aspergillus niger KLLA0D09999CAH00602.1 49642640 Kluyveromyces lactis

The conversion of an acid to an aldehyde is thermodynamicallyunfavorable and typically requires energy-rich cofactors and multipleenzymatic steps. For example, in butanol biosynthesis conversion ofbutyrate to butyraldehyde is catalyzed by activation of butyrate to itscorresponding acyl-CoA by a CoA transferase or ligase, followed byreduction to butyraldehyde by a CoA-dependent aldehyde dehydrogenase.Alternately, an acid can be activated to an acyl-phosphate andsubsequently reduced by a phosphate reductase. Direct conversion of theacid to aldehyde by a single enzyme is catalyzed by a bifunctionalcarboxylic acid reductase enzyme in the 1.2.1 family. Exemplary enzymesthat catalyze these transformations include carboxylic acid reductase,alpha-aminoadipate reductase and retinoic acid reductase.

Carboxylic acid reductase (CAR), found in Nocardia iowensis, catalyzesthe magnesium, ATP and NADPH-dependent reduction of carboxylic acids totheir corresponding aldehydes (Venkitasubramanian et al., J Biol. Chem.282:478-485 (2007)). The natural substrate of this enzyme is benzoicacid and the enzyme exhibits broad acceptance of aromatic and aliphaticsubstrates including fatty acids of length C12-C18 (Venkitasubramanianet al., Biocatalysis in Pharmaceutical and Biotechnology Industries. CRCpress (2006); WO 2010/135624). CAR requires post-translationalactivation by a phosphopantetheine transferase (PPTase) that convertsthe inactive apo-enzyme to the active holo-enzyme (Hansen et al., Appl.Environ. Microbiol 75:2765-2774 (2009)). The Nocardia CAR enzyme wascloned and functionally expressed in E. coli (Venkitasubramanian et al.,J Biol. Chem. 282:478-485 (2007)). Co-expression of the npt gene,encoding a specific PPTase, improved activity of the enzyme. A relatedenzyme from Mycobacterium sp. strain JLS catalyzes the reduction offatty acids of length C12-C16. Variants of this enzyme with enhancedactivity on fatty acids are described in WO 2010/135624.Alpha-aminoadipate reductase (AAR, EC 1.2.1.31), participates in lysinebiosynthesis pathways in some fungal species. This enzyme naturallyreduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. Thecarboxyl group is first activated through the ATP-dependent formation ofan adenylate that is then reduced by NAD(P)H to yield the aldehyde andAMP. Like CAR, this enzyme utilizes magnesium and requires activation bya PPTase. Enzyme candidates for AAR and its corresponding PPTase arefound in Saccharomyces cerevisiae (Morris et al., Gene 98:141-145(1991)), Candida albicans (Guo et al., Mol. Genet. Genomics 269:271-279(2003)), and Schizosaccharomyces pombe (Ford et al., Curr. Genet.28:131-137 (1995)). The AAR from S. pombe exhibited significant activitywhen expressed in E. coli (Guo et al., Yeast 21:1279-1288 (2004)). TheAAR from Penicillium chrysogenum accepts S-carboxymethyl-L-cysteine asan alternate substrate, but did not react with adipate, L-glutamate ordiaminopimelate (Hijarrubia et al., J Biol. Chem. 278:8250-8256 (2003)).The gene encoding the P. chrysogenum PPTase has not been identified todate and no high-confidence hits were identified by sequence comparisonhomology searching.

Protein GenBank ID GI Number Organism car AAR91681.1 40796035 Nocardiaiowensis npt ABI83656.1 114848891 Nocardia iowensis car YP_001070587.1126434896 Mycobacterium sp. strain JLS npt YP_001070355.1 126434664Mycobacterium sp. strain JLS LYS2 AAA34747.1 171867 Saccharomycescerevisiae LYS5 P50113.1 1708896 Saccharomyces cerevisiae LYS2AAC02241.1 2853226 Candida albicans LYS5 AAO26020.1 28136195 Candidaalbicans Lys1p P40976.3 13124791 Schizosaccharomyces pombe Lys7pQ10474.1 1723561 Schizosaccharomyces pombe Lys2 CAA74300.1 3282044Penicillium chrysogenum

Additional car and npt genes can be identified based on sequencehomology.

GenBank Gene name GI No. Accession No. Organism fadD9 121638475YP_978699.1 Mycobacterium bovis BCG BCG_2812c 121638674 YP_978898.1Mycobacterium bovis BCG nfa20150 54023983 YP_118225.1 Nocardia farcinicaIFM 10152 nfa40540 54026024 YP_120266.1 Nocardia farcinica IFM 10152SGR_6790 YP_001828302.1 182440583 Streptomyces griseus subsp. griseusNBRC 13350 SGR_665 YP_001822177.1 182434458 Streptomyces griseus subsp.griseus NBRC 13350 MSMEG_2956 YP_887275.1 118473501 Mycobacteriumsmegmatis MC2 155 MSMEG_5739 YP_889972.1 118469671 Mycobacteriumsmegmatis MC2 155 MSMEG_2648 YP_886985.1 118471293 Mycobacteriumsmegmatis MC2 155 MAP1040c NP_959974.1 41407138 Mycobacterium aviumsubsp. paratuberculosis K-10 MAP2899c NP_961833.1 41408997 Mycobacteriumavium subsp. paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131Mycobacterium marinum M MMAR_2936 YP_001851230.1 183982939 Mycobacteriummarinum M MMAR_1916 YP_001850220.1 183981929 Mycobacterium marinum MTpau_1373 YP_003646340.1 296139097 Tsukamurella paurometabola DSM 20162Tpau_1726 YP_003646683.1 296139440 Tsukamurella paurometabola DSM 20162CPCC7001_1320 ZP_05045132.1 254431429 Cyanobium PCC7001 DDBDRAFT_0187729XP_636931.1 66806417 Dictyostelium discoideum AX4

An additional enzyme candidate found in Streptomyces griseus is encodedby the griC and griD genes. This enzyme is believed to convert3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde asdeletion of either griC or griD led to accumulation of extracellular3-acetylamino-4-hydroxybenzoic acid, a shunt product of3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot.60(6):380-387 (2007)). Co-expression of griC and griD with SGR_(—)665,an enzyme similar in sequence to the Nocardia iowensis npt, can bebeneficial.

GenBank Gene name GI No. Accession No. Organism griC YP_001825755.1182438036 Streptomyces griseus subsp. griseus NBRC 13350 griDYP_001825756.1 182438037 Streptomyces griseus subsp. griseus NBRC 13350

Example X Production of 1,3-Butanediol from Glucose in Saccharomycescerevisiae

This example illustrates the construction and biosynthetic production of1,3-BDO from glucose in Saccharomyces cerevisiae.

The pathway for 1,3-BDO production is comprised of two MI-FAE cycleenzymes (thiolase and 3-oxoacyl-CoA reductase), in conjunction withtermination pathway enzymes (acyl-CoA reductase (aldehyde forming) andalcohol dehydrogenase). The 1,3-BDO pathway engineered into S.cerevisiae is composed of four enzymatic steps which transformacetyl-CoA to 1,3-BDO. The first step entails the condensation of twomolecules of acetyl-CoA into acetoacetyl-CoA by an acetoacetyl-CoAthiolase enzyme (THL). In the second step, acetoacetyl-CoA is reduced to3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase, also called3-hydroxybutyryl-CoA dehydrogenase (HBD). 3-hydroxybutyryl-CoA reductase(ALD) catalyzes formation of the aldehyde from the acyl-CoA. Furtherreduction of 3-hydroxybutyraldehyde to 1,3-BDO is catalyzed by 1,3-BDOdehydrogenase (ADH).

To enable 13-BDO production in the cytosol, two acetyl-CoA formingpathways were engineered into S. cerevisiae. The first pathway entailsconversion of pyruvate to acetyl-CoA by pyruvate decarboxylase (FIG.2E), acetaldehyde dehydrogenase (FIG. 2F) and acetyl-CoA synthetase(FIG. 2B). The second pathway is pyruvate formate lyase (FIG. 2H).

For each enzymatic step of the 1,3-BDO pathway, a list of applicablegenes was assembled for corroboration. The genes cloned and assessed inthis study are presented below in Table 1, along with the appropriatereferences and URL citations to the polypeptide sequence.

TABLE 1 Exemplary NCBI Step ID Gene Accession # GI Source OrganismAcetoacetyl-CoA thiolase (THL) FIG. 1A 1502 thiI P45359.1 1174677Clostridium acetobutylicum ATCC 824 FIG. 1A 1491 atoB NP_416728 16130161Escherichia coli str. K-12 substr. MG1655 FIG. 1A 560 thiA NP_349476.115896127 Clostridium acetobutylicum ATCC 824 FIG. 1A 1512 phbA P07097.4135759 Zoogloea ramigera FIG. 1A 1501 phbA P14611.1 135754 Ralstoniaeutropha H16 3-Hydroxybutyryl-CoA dehydrogenase (HBD) FIG. 1B 1495 hbdAAM14586.1 20162442 Clostridium beijerinckii NCIMB 80523-Hydroxybutyryl-CoA reductase (ALD) FIG. 1E 707 Lvis_1603 YP_795711.1116334184 Lactobacillus brevis ATCC 367 3-Hydroxybutyraldehyde reductase(ADH) FIG. 1F 28 bdh BAF45463.1 124221917 Clostridiumsaccharoperbutylacetonicum Pyruvate formate lyase (PflAB) FIG. 2H 1799pflA NP_415422.1 16128869 Escherichia coli MG1655 FIG. 2H 500 pflBNP_415423 16128870 Escherichia coli MG1655 PDH Bypass (aldehydedehydrogenase, acetyl-CoA synthase) FIG. 2F 1849 ALD6 NP_015264.16325196 Saccharomyces cerevisiae S288c FIG. 2B 1845 Acs AAL23099.116422835 Salmonella enterica LT2 FIG. 2B 1845A Acsm AAL23099.1 16422835Salmonella enterica LT2

Genes were cloned via PCR from the genomic DNA of the native orwild-type organism. Primers used to amplify the pathway genes are (from5′ to 3; underlined sequences are gene specific):

Thl 1502: FP: (SEQ ID NO:)TCTAATCTAAGTTTTCTAGAACTAGTAAAGATGAGAGATGTAGTAATA GTAAGTGCTGTA RP:(SEQ ID NO:) GATATCGAATTCCTGCAGCCCGGGGGATCCTTAGTCTCTTTCAACTACG AGAGCTGTTThl 1491: FP: (SEQ ID NO: 11)TCTAATCTAAGTTTTCTAGAACTAGTAAAGATGAAAAATTGTGTCATCG TCAGTG RP:(SEQ ID NO:) GATATCGAATTCCTGCAGCCCGGGGGATCCTTAATTCAACCGTTCAATCACCATCGCAAT Thl 560: FP: (SEQ ID NO:)AATCTAAGTTTTCTAGAACTAGTAAAGATGAAAGAAGTTGTAATAGCT AGTGCAGTAA RP:(SEQ ID NO:) TATCGAATTCCTGCAGCCCGGGGGATCCTTAATGGTGATGGTGATGATGGCACTTTTCTA Thl 1512: FP: (SEQ ID NO:)TCTAATCTAAGTTTTCTAGAACTAGTAAAGATGAGCACCCCGTCCATCG TCA PR: (SEQ ID NO:)GATATCGAATTCCTGCAGCCCGGGGGATCCCTAAAGGCTCTCGATGCA CATCGCC Thl 1501: FP:(SEQ ID NO:) TAAGCTAGCAAGAGGAGAAGTCGACATGACTGACGTTGTCATCGTATC CGC RP:(SEQ ID NO:) GCCTCTAGGAAGCTTTCTAGATTATTATTTGCGCTCGACTGCCAGC Hbd 1495:FP: (SEQ ID NO:) AAGCATACAATCAACTATCTCATATACAATGAAAAAGATTTTTGTACTTGGAGCA RP: (SEQ ID NO:) AAAAATCATAAATCATAAGAAATTCGCTTATTTAGAGTAATCATAGAATCCTTTTCCTGA Ald 707: FP: (SEQ ID NO:)AATCTAAGTTTTCTAGAACTAGTAAAGATGAACACAGAAAACATTGAA CAAGCCAT RP:(SEQ ID NO:) TATCGAATTCCTGCAGCCCGGGGGATCCCTAAGCCTCCCAAGTCCGTAATGAGAACCCTT Adh 28: FP: (SEQ ID NO:)CCAAGCATACAATCAACTATCTCATATACAATGGAGAATTTTAGATTTA ATGCATATACA RP:(SEQ ID NO:) AATAAAAATCATAAATCATAAGAAATTCGCTTAAAGGGACATTTCTAAAATTTTATATAC

1845A is a sequence variant of the wild type (1845) enzyme. Thevariation is a point mutation in the residue Leu-641 (L641P), describedin Starai and coworkers (Starai et al, J Biol Chem 280: 26200-5 (2005)).The function of the mutation, e.g., is to prevent post-translationalregulation by acetylation and maintain the Acs enzyme in its activestate.

Shuttle plasmids shown in Table 2 were constructed for expression ofheterologous genes in S. cerevisiae. Plasmids d9, d10, and d11 are emptyplasmid controls with the selection marker of Ura, His, and Leu,respectively. Plasmids d12 or d13 contains a single ALD or ADH gene withthe URA3 selection marker. Plasmids d14, d16, and d17 contains hbd andthil genes with the HIS3 selection marker.

TABLE 2 Plasmid Selection Marker Gene(s) pESC-L URA3 NA pESC-H HIS3 NApESC-U LEU2 NA pY3Hd1 URA3 1799(pflA)-500(pflB) pY3Hd2 HIS31799(pflA)-500(pflB) pY3Hd3 LEU2 1799(pflA)-500(pflB) pY3Hd4 URA31849(ALD6)-1845(Acs) pY3Hd5 URA3 1849(ALD6)-1845A(Acsm) pY3Hd6 URA31495(Hbd)-1491(Thl) pY3Hd7 URA3 1495(Hbd)-560(Thl) pY3Hd8 LEU228(ADH)-707(ALD) pY3Hd9 URA3 NA pY3Hd10 HIS3 NA pY3Hd11 LEU2 NA pY3Hd12URA3 707(ALD) pY3Hd13 URA3 28(ADH) pY3Hd14 HIS3 1495(Hbd)-1502(Thl)pY3Hd15 HIS3 1495(Hbd)-1512(Thl) pY3Hd16 HIS3 1495(Hbd)-1491(Thl)pY3Hd17 HIS3 1495(Hbd)-560(Thl)

Yeast host BY4741 [MATa his3Δ0 leu2Δ0 met15Δ0 ura3Δ0] was chosen as thehost strain for this work as a wild-type laboratory strain with theappropriate auxotrophic markers to host the pathway plasmids. BY4741 wastransformed with plasmids containing 1,3-BDO pathway genes alone oralong with plasmids that contain PDH bypass genes or pflAB genes. Vectorbackbones used in this example include p427TEF yeast expression vectors,the pY3H bridging vectors (Sunrise Science) and pESC yeast epitopetagging vectors (Agilent Technologies). The pY3H vector containing aTEF1 promoter, CYC terminator and URA3 selection marker from S.cerevisiae was used to build dual-promoter plasmids with differentselection markers. ADH1 promoter and terminator sequences from S.cerevisiae were inserted upstream of the TEF1 promoter so the twotranscriptional units are in a back-to-back orientation. The SV40nuclear localization signal sequence was removed during the cloningprocess. The resulting plasmid was named pY3Hd9. To construct plasmidswith a different selection marker, the URA3 gene in pY3Hd9 was replacedwith the HIS3 or LEU2 gene from S. cerevisiae to produce pY3Hd10 andpY3Hd11, respectively. Two of the four 1,3-BDO pathway genes—Hbd and Th1(see Table 103 for gene numbers)—were cloned into the dual-promoterplasmid with the HIS3 marker such that the expression of the Hbd genesis controlled by the ADH1 promoter while the expression of the Th1 geneis controlled by the TEF1 promoter (pY3Hd14˜17). Ald and Adh genes werecloned into the dual-promoter plasmid with the LEU2 selection markersuch that the ADH1 promoter drives the adh genes and the TEF1 promoterdrives the ald genes (pY3Hd8). The PflAB genes or the PDH bypass genes(ALD6 and acs) were cloned into the dual-promoter plasmid with the URA3marker where pflA or ALD6 is controlled under the ADH1 promoter and pflBor acs is controlled under the TEF1 promoter. Yeast transformation wasdone using Frozen-EZ Yeast Transformation (Zymo Research).

Tables 3 and 4 show the combinations of plasmids and experimentalconditions tested.

TABLE 3 Sample Plasmid 1 Plasmid 2 plasmid 3 gene 1 gene 2 gene 3 gene 4gene 5 gene 6 Aeroation Note 1 pESC-L pESG-H Anaerobic EV2 2 pESC-LpESC-H 23G EV2 3 d8 d16 1495 1491 28 707 Anaerobic BDO 4 d8 d16 14951491 28 707 Anaerobic BDO 5 d8 d16 1495 1491 28 707 23G BDO 6 d8 d161495 1491 28 707 23G BDO 7 d8 d17 1495 560 28 707 Anaerobic BDO 8 d8 d171495 560 28 707 Anaerobic BDO 9 d8 d17 1495 560 28 707 23G BDO 10 d8 d171495 560 28 707 23G BDO 11 pESC-H pESC-L pESC-U Anaerobic EV3 12 pESC-HpESC-L pESC-U 23G EV3 13 d8 d16 d1 1495 1491 28 707 pflA pflB AnaerobicBDO + pflAB 14 d8 d16 d1 1495 1491 28 707 pflA pflB Anaerobic BDO +pflAB 15 d8 d16 d1 1495 1491 28 707 pflA pflB 23G BDO + pflAB 16 d8 d16d1 1495 1491 28 707 pflA pflB 23G BDO + pflAB 17 d8 d17 d1 1495 560 28707 pflA pfIE Anaerobic BDO + pflAB 18 d8 d17 d1 1495 560 28 707 pflApflB Anaerobic BDO + pflAB 19 d8 d17 d1 1495 560 28 707 pflA pflB 23GBDO + pflAB 20 d8 d17 d1 1495 560 28 707 pflA pfIE 23G BDO + pflAB 21 d8d16 d5 1495 1491 28 707 ALD6 acsm Anaerobic EDO + PDH 22 d8 d16 d5 14951491 28 707 ALD6 acsm Anaerobic EDO + PDH 23 d8 d16 d5 1495 1491 28 707ALD6 acsm 23G EDO + PDH 24 d8 d16 d5 1495 1491 28 707 ALD6 acsm 23GEDO + PDH 25 d8 d17 d5 1495 560 28 707 ALD6 acsm Anaerobic EDO + PDH 26d8 d17 d5 1495 560 28 707 ALD6 acsm Anaerobic EDO + PDH 27 d8 d17 d51495 560 28 707 ALD6 acsm 23G EDO + PDH 28 d8 d17 d5 1495 560 28 707ALD6 acsm 23G EDO + PDH

TABLE 4 Plasmid 1 Plasmid 2 plasmid 3 gene 1 gene 2 gene 3 gene 4 gene 5gene 6 Aeroation Note d9 d11 aerobic EVC d8 d17 1495 560 28 707 aerobicBDO d8 d17 d5 1495 560 28 707 1849 1845A aerobic BDO + PDH d8 d14 14951502 28 707 aerobic BDO d8 d14 d5 1495 1502 28 707 1849 1845A aerobicBDO + PDH

In Table 3, colonies were inoculated in 5 ml of 2% glucose medium withcorresponding amino acid dropouts and cultured at 30 degree forapproximately 48 hrs. Cells were briefly spun down and re-suspended in 2ml fresh 2% glucose medium with tween-80 and ergosterol added.Resuspended cultures were added to 10 ml fresh glucose medium in 20 mlbottles to obtain a starting OD of 0.2. For anaerobic cultures, thebottles containing cultures were vacuumed and filled with nitrogen. Formicro-aerobic growth, a 23G needle was inserted. All the cultures wereincubated at 30 degree with shaking for 24 hours. In Table 4, theexperiment was carried out in a 96-well plate and cells grownaerobically in 1.2 ml of medium with varying glucose and acetateconcentrations (5% glucose, 10% glucose, 5% glucose+50 mM acetate, and10% glucose+50 mM acetate).

Concentrations of glucose, 1,3-BDO, alcohols, and other organic acidbyproducts in the culture supernatant were determined by HPLC using anHPX-87H column (BioRad).

MI-FAE cycle and termination pathway genes were tested with or withoutpflAB or PDH bypass. As shown in FIGS. 9-11, these constructs produced0.3-3.35 mM 1,3-BDO in yeast S. cerevisiae BY4741, and ethanol wasproduced in the tested samples tested. The PDH bypass (here,overexpression of ALD6 and acs or acsm genes) improved production of1,3-BDO.

Example XI Enzymatic Activity of 1,3-Butanediol Pathway Enzymes

This example describes the detection of 1,3-BDO pathway enzyme activityusing in vitro assays.

Activity of the heterologous enzymes was tested in in vitro assays,using an internal yeast strain as the host for the plasmid constructscontaining the pathway genes. Cells were grown aerobically in yeastmedia containing the appropriate amino acid for each construct. Toobtain crude extracts for activity assays, cells were harvested bycentrifugation. The pellets were resuspended in 0.1 mL 100 mM Tris pH7.0 buffer containing protease inhibitor cocktail. Lysates were preparedusing the method of bead beating for 3 min. Following bead beating, thesolution was centrifuged at 14,000 rpm (Eppendorf centrifuge 5402) for15 min at 4° C. Cell protein in the sample was determined using themethod of Bradford et al., Anal. Biochem. 72:248-254 (1976), andspecific enzyme assays conducted as described below.

Thiolase

Thiolase enzymes catalyze the condensation of two acetyl-CoA to formacetoacetyl-CoA. In the reaction, coenzyme A (CoA) is released and thefree CoA can be detected using 5,5′-dithiobis-2-nitrobenzoic acid (DTNB)which absorbs at 410 nm upon reaction with CoA. Five thiolases weretested (see example X, Table 1). Estimated specific activity in E. colicrude lysates is shown in FIG. 12.

Among the Th1 that showed expressed protein, 1512 and 1502 demonstratedthe highest specific activity for acetyl-CoA condensation activity n E.coli crude lysates.

Both 1491 and 560 were cloned in dual promoter yeast vectors with 1495,which is the 3-hydroxybutyryl-CoA dehydrogenase (see FIG. 13). Thesethiolases were evaluated for acetyl-CoA condensation activity, and thedata is shown in FIG. 13. The results indicate that both 560 and 1491demonstrate an initial burst of activity that is too fast to measure.However, after the initial enzyme rate, the condensation rate of 560 isgreater than 1491. Thus, there is protein expression and active enzymewith the yeast dual promoter vectors as indicated by active thiolaseactivity observed in crude lysates.

3-Hydroxybutyryl-CoA Dehydrogenase (Hbd)

Acetoacetyl-CoA is metabolized to 3-hydroxybutyryl-CoA by3-hydroxybutyryl-CoA dehydrogenase. The reaction requires oxidation ofNADH, which can be monitored by fluorescence at an excitation wavelengthat 340 nm and an emission at 460 nm. The oxidized form, NAD+, does notfluoresce. This detection strategy was used for all of the dehydrogenasesteps. 1495, the Hbd from Clostridium beijerinckii, was assayed in thedual promoter yeast vectors that contained either 1491 (vectorid=pY3Hd17) or 560 (vector id=pY3Hd16). See Table 1 for GenBankidentifiers of each enzyme. The time course data is shown in FIG. 14.

The Hbd rate of 1495 containing 560 was much faster than 1491. Theresults provided in FIG. 15 show that the Hbd prefers NADH over NADPH.The Hbd enzyme appears to display the fastest catalytic activity amongthe four pathway enzymes in crude lysates. The Hbd enzyme, i.e. a3-ketoacyl-CoA reductase, is an example of a MI-FAE cycle or MD-FAEcycle enzyme that preferentially reacts with an NADH cofactor.

Aldehyde Deyhdrogenase (Ald)

An aldehyde reductase converts 3-hydroxybutyryl-CoA to3-hydroxybutyraldehyde. This reaction requires NAD(P)H oxidation, whichcan be used to monitor enzyme activity. The Ald from Lactobacillusbrevis (Gene ID 707) was cloned in a dual vector that contained thealcohol dehydrogenase from Clostridium saccharoperbutylacetonicum (GeneID 28). These two enzymes were cloned in another dual promoter yeastvector containing a Leu marker.

The Ald activity data for crude lysates is shown in FIG. 16 with a 707lysate from E. coli used as a standard. The results indicate the 707showed enzyme activity in yeast lysates that is comparable to the lysatefrom bacteria. In addition, the 707 gene product prefers NADH to NADPHas the cofactor. The 707 gene product, i.e. an acy-CoA reductase(aldehyde forming), is an example of a termination pathway enzyme thatpreferentially reacts with an NADH cofactor.

Alcohol Dehydrogenase (Adh)

1,3-BDO is formed by an alcohol dehydrogenase (Adh), which reduces3-hydroxybutyraldehyde in the presence of NAD(P)H. The oxidation ofNAD(P)H can be used to monitor the reaction as described above.

The evaluation of ADH (Gene 28) in the dual promoter vector with ALD(Gene 707) is shown in FIG. 17 with butyraldehyde, a surrogate substratefor 3-hydroxybutyraldehyde. The data indicate that Gene 28 have Adhactivity similar to the no insert control (EV) with butyraldehyde andNADPH. This is likely caused by endogenous ADH enzymes present in yeastthat may function in the same capability as 28.

In summary, candidates for the Thl, Hbd, Ald, and Adh to produce 1,3-BDOshowed enzyme activity in yeast crude lysates for the dual promotervectors constructed.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples provided above, it should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

What is claimed is:
 1. A non-naturally occurring microbial organismhaving a malonyl-CoA independent fatty acyl-CoA elongation (MI-FAE)cycle and/or a malonyl-CoA dependent fatty acyl-CoA elongation (MD-FAE)cycle in combination with a termination pathway, wherein said MI-FAEcycle comprises one or more thiolase, one or more 3-oxoacyl-CoAreductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or moreenoyl-CoA reductase, wherein said MD-FAE cycle comprises one or moreelongase, one or more 3-oxoacyl-CoA reductase, one or more3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase,wherein said termination pathway comprises a pathway selected from: (1)1H; (2) 1K and 1L; (3) 1E and 1N; (4) 1K, 1J, and 1N; (5) 1E; (6) 1K and1J; (7) 1H and 1N; (8) 1K, 1L, and 1N; (9) 1E and 1F; (10) 1K, 1J, and1F; (11) 1H, 1N, and 1F; (12) 1K, 1L, 1N, and 1F; and (13) 1G, wherein1E is an acyl-CoA reductase (aldehyde forming), wherein 1F is an alcoholdehydrogenase, wherein 1G is an acyl-CoA reductase (alcohol forming),wherein 1H is an acyl-CoA hydrolase, acyl-CoA transferase or acyl-CoAsynthase, wherein 1J is an acyl-ACP reductase, wherein 1K is anacyl-CoA:ACP acyltransferase, wherein 1L is a thioesterase, wherein 1Nis an aldehyde dehydrogenase (acid forming) or a carboxylic acidreductase, wherein an enzyme of the MI-FAE cycle, MD-FAE cycle ortermination pathway is encoded by at least one exogenous nucleic acidand is expressed in a sufficient amount to produce a compound of Formula(I):

wherein R₁ is C₁₋₂₄ linear alkyl; R₂ is CH₂OH, CHO, or COOH; R₃ is H,OH, or oxo (═O); and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four, wherein the substrateof each of said enzymes of the MI-FAE cycle, MD-FAE cycle and thetermination pathway are independently selected from a compound ofFormula (II), malonyl-CoA, propionyl-CoA or acetyl-CoA:

wherein R₁ is C₁₋₂₄ linear alkyl; R₃ is H, OH, or oxo (═O); R₄ is S-CoA,ACP, OH or H; and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four; wherein said one ormore enzymes of the MI-FAE cycle are each selective for a compound ofFormula (II) having a number of carbon atoms at R₁ that is no greaterthan the number of carbon atoms at R₁ of said compound of Formula (I),wherein said one or more enzymes of the MD-FAE cycle are each selectivefor a compound of Formula (II) having a number of carbon atoms at R₁that is no greater than the number of carbon atoms at R₁ of saidcompound of Formula (I), and wherein said one or more enzymes of thetermination pathway are each selective for a compound of Formula (II)having a number of carbon atoms at R₁ that is no less than the number ofcarbon atoms at R₁ of said compound of Formula (I).
 2. The non-naturallyoccurring microbial organism of claim 1, wherein R₁ is C₁₋₁₇ linearalkyl.
 3. The non-naturally occurring microbial organism of claim 2,wherein R₁ is C₉ linear alkyl, C₁₀ linear alkyl, C₁₁, linear alkyl, C₁₂linear alkyl or C₁₃ linear alkyl.
 4. The non-naturally occurringmicrobial organism of claim 1, wherein said microbial organism comprisestwo, three, or four exogenous nucleic acids each encoding an enzyme ofsaid MI-FAE cycle or said MD-FAE cycle.
 5. The non-naturally occurringmicrobial organism of claim 1, wherein said microbial organism comprisestwo, three, or four exogenous nucleic acids each encoding an enzyme ofsaid termination pathway.
 6. The non-naturally occurring microbialorganism of claim 3, wherein said microbial organism comprises exogenousnucleic acids encoding each of the enzymes of at least one of thepathways selected from (1)-(13).
 7. The non-naturally occurringmicrobial organism of claim 1, wherein said at least one exogenousnucleic acid is a heterologous nucleic acid.
 8. The non-naturallyoccurring microbial organism of claim 1, wherein said non-naturallyoccurring microbial organism is in a substantially anaerobic culturemedium.
 9. The non naturally occurring microbial organism of claim 1,wherein said enzyme of the MI-FAE cycle, MD-FAE cycle or terminationpathway is expressed in a sufficient amount to produce a compoundselected from the Formulas (III)-(VI):

wherein R₁ is C₁₋₁₇ linear alkyl.
 10. The non-naturally occurringmicrobial organism of claim 9, wherein R₁ is C₉ linear alkyl, C₁₀ linearalkyl, C₁₁, linear alkyl, C₁₂ linear alkyl or C₁₃ linear alkyl.
 11. Thenon-naturally occurring microbial organism of claim 1, wherein saidmicrobial organism further comprises an acetyl-CoA pathway and at leastone exogenous nucleic acid encoding an acetyl-CoA pathway enzymeexpressed in a sufficient amount to produce acetyl-CoA, wherein saidacetyl-CoA pathway comprises a pathway selected from: (1) 2A and 2B; (2)2A, 2C, and 2D; (3) 2H; (4) 2G and 2D; (5) 2E, 2F and 2B; (6) 2E and 2I;(7) 2J, 2F and 2B; (8) 2J and 2I; (9) 3A, 3B, and 3C; (10) 3A, 3B, 3J,3K, and 3D; (11) 3A, 3B, 3G, and 3D; (12) 3A, 3F, and 3D; (13) 3N, 3H,3B and 3C; (14) 3N, 3H, 3B, 3J, 3K, and 3D; (15) 3N, 3H, 3B, 3G, and 3D;(16) 3N, 3H, 3F, and 3D; (17) 3L, 3M, 3B and 3C; (18) 3L, 3M, 3B, 3J,3K, and 3D; (19) 3L, 3M, 3B, 3G, and 3D; (20) 3L, 3M, 3F, and 3D; (21)4A, 4B, 4D, 4H, 4I, and 4J; (22) 4A, 4B, 4E, 4F, 4H, 4I, and 4J; (23)4A, 4B, 4E, 4K, 4L, 4H, 4I, and 4J; (24) 4A, 4C, 4D, 4H, and 4J; (25)4A, 4C, 4E, 4F, 4H, and 4J; (26) 4A, 4C, 4E, 4K, 4L, 4H, and 4J; (27)5A, 5B, 5D, and 5G; (28) 5A, 5B, 5E, 5F, and 5G; (29) 5A, 5B, 5E, 5K,5L, and 5G; (30) 5A, 5C, and 5D; (31) 5A, 5C, 5E, and 5F; and (32) 5A,5C, 5E, 5K, and 5L, wherein 2A is a pyruvate oxidase (acetate-forming),wherein 2B is an acetyl-CoA synthetase, an acetyl-CoA ligase or anacetyl-CoA transferase, wherein 2C is an acetate kinase, wherein 2D is aphosphotransacetylase, wherein 2E is a pyruvate decarboxylase, wherein2F is an acetaldehyde dehydrogenase, wherein 2G is a pyruvate oxidase(acetyl-phosphate forming), wherein 2H is a pyruvate dehydrogenase, apyruvate:ferredoxin oxidoreductase, a pyruvate:NAD(P)H oxidoreductase ora pyruvate formate lyase, wherein 2I is an acetaldehyde dehydrogenase(acylating), wherein 2J is a threonine aldolase, wherein 3A is aphosphoenolpyruvate (PEP) carboxylase or a PEP carboxykinase, wherein 3Bis an oxaloacetate decarboxylase, wherein 3C is a malonate semialdehydedehydrogenase (acetylating), wherein 3D is an acetyl-CoA carboxylase ora malonyl-CoA decarboxylase, wherein 3F is an oxaloacetate dehydrogenaseor an oxaloacetate oxidoreductase, wherein 3G is a malonate semialdehydedehydrogenase (acylating), wherein 3H is a pyruvate carboxylase, wherein3J is a malonate semialdehyde dehydrogenase, wherein 3K is a malonyl-CoAsynthetase or a malonyl-CoA transferase, wherein 3L is a malic enzyme,wherein 3M is a malate dehydrogenase or a malate oxidoreductase, wherein3N is a pyruvate kinase or a PEP phosphatase, wherein 4A is a citratesynthase, wherein 4B is a citrate transporter, wherein 4C is acitrate/malate transporter, wherein 4D is an ATP citrate lyase, wherein4E is a citrate lyase, wherein 4F is an acetyl-CoA synthetase or anacetyl-CoA transferase, wherein 4H is a cytosolic malate dehydrogenase,wherein 4I is a malate transporter, wherein 4J is a mitochondrial malatedehydrogenase, wherein 4K is an acetate kinase, wherein 4L is aphosphotransacetylase, wherein 5A is a citrate synthase, wherein 5B is acitrate transporter, wherein 5C is a citrate/oxaloacetate transporter,wherein 5D is an ATP citrate lyase, wherein 5E is a citrate lyase,wherein 5F is an acetyl-CoA synthetase or an acetyl-CoA transferase,wherein 5G is an oxaloacetate transporter, wherein 5K is an acetatekinase, and wherein 5L is a phosphotransacetylase.
 12. The non-naturallyoccurring microbial organism of claim 11, wherein said microbialorganism comprises two, three, four, five, six, seven or eight exogenousnucleic acids each encoding an acetyl-CoA pathway enzyme.
 13. Thenon-naturally occurring microbial organism of claim 12, wherein saidmicrobial organism comprises exogenous nucleic acids encoding each ofthe acetyl-CoA pathway enzymes of at least one of the pathways selectedfrom (1)-(32).
 14. The non-naturally occurring microbial organism ofclaim 1, further comprising one or more gene disruptions, said one ormore gene disruptions occurring in endogenous genes encoding proteins orenzymes involved in: native production of ethanol, glycerol, acetate,formate, lactate, CO₂, fatty acids, or malonyl-CoA by said microbialorganism; transfer of pathway intermediates to cellular compartmentsother than the cytosol; or native degradation of a MI-FAE cycleintermediate, MD-FAE cycle intermediate or a termination pathwayintermediate by said microbial organism, wherein said one or more genedisruptions confer increased production of the compound of Formula (I)in said microbial organism.
 15. The non-naturally occurring microbialorganism of claim 14, wherein said protein or enzyme is selected fromthe group consisting of a fatty acid synthase, an acetyl-CoAcarboxylase, a biotin:apoenzyme ligase, an acyl carrier protein, athioesterase, an acyltransferase, an ACP malonyltransferase, a fattyacid elongase, an acyl-CoA synthetase, an acyl-CoA transferase, anacyl-CoA hydrolase, a pyruvate decarboxylase, a lactate dehydrogenase,an alcohol dehydrogenase, an acid-forming aldehyde dehydrogenases, anacetate kinase, a phosphotransacetylase, a pyruvate oxidase, aglycerol-3-phosphate dehydrogenase, a glycerol-3-phosphate phosphatase,a mitochondrial pyruvate carrier, a peroxisomal fatty acid transporter,a peroxisomal acyl-CoA transporter, a peroxisomalcarnitine/acylcarnitine transferase, an acyl-CoA oxidase, and anacyl-CoA binding protein.
 16. The non-naturally occurring microbialorganism of claim 1, wherein one or more enzymes of the MI-FAE cycle,MD-FAE cycle or the termination pathway preferentially react with anNADH cofactor or have reduced preference for reacting with an NAD(P)Hcofactor, wherein said one or more enzymes of the MI-FAE cycle or MD-FAEcycle are a 3-ketoacyl-CoA reductase or an enoyl-CoA reductase, andwherein said one or more enzymes of the termination pathway are selectedfrom an acyl-CoA reductase (aldehyde forming), an alcohol dehydrogenase,an acyl-CoA reductase (alcohol forming), an aldehyde decarbonylase, anacyl-ACP reductase, an aldehyde dehydrogenase (acid forming) and acarboxylic acid reductase.
 17. The non-naturally occurring microbialorganism of claim 1, further comprising one or more gene disruptions,said one or more gene disruptions occurring in genes encoding proteinsor enzymes that result in an increased ratio of NAD(P)H to NAD(P)present in the cytosol of said microbial organism following saiddisruptions.
 18. The non-naturally occurring microbial organism of claim17, wherein said gene encoding a protein or enzyme that results in anincreased ratio of NAD(P)H to NAD(P) present in the cytosol of saidmicrobial organism following said disruptions is selected from the groupconsisting of an NADH dehydrogenase, a cytochrome oxidase, aglycerol-3-phosphate dehydrogenase, glycerol-3-phosphate phosphatase, analcohol dehydrogenase, a pyruvate decarboxylase, an aldehydedehydrogenase (acid forming), a lactate dehydrogenase, aglycerol-3-phosphate dehydrogenase, a glycerol-3-phosphate:quinoneoxidoreductase, a malic enzyme and a malate dehydrogenase.
 19. Thenon-naturally occurring organism of claim 14 or 17, wherein said one ormore gene disruptions comprises a deletion of said one or more genes.20. The non-naturally occurring microbial organism of claim 1, whereinsaid microbial organism is Crabtree positive and is in culture mediumcomprising excess glucose, thereby increasing the ratio of NAD(P)H toNAD(P) present in the cytosol of said microbial organism.
 21. Thenon-naturally occurring microbial organism of claim 1, furthercomprising at least one exogenous nucleic acid encoding an extracellulartransporter or an extracellular transport system for the compound ofFormula (I).
 22. The non-naturally occurring microbial organism of claim1, wherein one or more endogenous enzymes involved in: native productionof ethanol, glycerol, acetate, formate, lactate, CO2, fatty acids, ormalonyl-CoA by said microbial organism; transfer of pathwayintermediates to cellular compartments other than the cytosol; or nativedegradation of a MI-FAE cycle intermediate, MD-FAE cycle intermediate ora termination pathway intermediate by said microbial organism, hasattenuated enzyme activity or expression levels.
 23. The non-naturallyoccurring microbial organism of claim 22, wherein said enzyme isselected from the group consisting of a fatty acid synthase, anacetyl-CoA carboxylase, a biotin:apoenzyme ligase, a thioesterase, anacyl carrier protein, a thioesterase, an acyltransferase, an ACPmalonyltransferase, a fatty acid elongase, an acyl-CoA synthetase, anacyl-CoA transferase, an acyl-CoA hydrolase, a pyruvate decarboxylase, alactate dehydrogenase, a short-chain alcohol dehydrogenase, anacid-forming aldehyde dehydrogenase, an acetate kinase, aphosphotransacetylase, a pyruvate oxidase, a glycerol-3-phosphatedehydrogenase, a glycerol-3-phosphate phosphatase, a mitochondrialpyruvate carrier, a peroxisomal fatty acid transporter, a peroxisomalacyl-CoA transporter, a peroxisomal carnitine/acylcarnitine transferase,an acyl-CoA oxidase, and an acyl-CoA binding protein.
 24. Thenon-naturally occurring microbial organism of claim 1, wherein one ormore endogenous enzymes involved in the oxidation of NAD(P)H or NADH,has attenuated enzyme activity or expression levels.
 25. Thenon-naturally occurring microbial organism of claim 24, wherein said oneor more endogenous enzymes are selected from the group consisting of anNADH dehydrogenase, a cytochrome oxidase, a glycerol-3-phosphatedehydrogenase, glycerol-3-phosphate phosphatase, an alcoholdehydrogenase, a pyruvate decarboxylase, an aldehyde dehydrogenase (acidforming), a lactate dehydrogenase, a glycerol-3-phosphate dehydrogenase,a glycerol-3-phosphate:quinone oxidoreductase, a malic enzyme and amalate dehydrogenase.
 26. A method for producing a compound of Formula(I):

wherein R₁ is C₁₋₂₄ linear alkyl; R₂ is CH₂OH, CHO, or COOH; R₃ is H,OH, or oxo (═O); and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four, comprising culturingthe non-naturally occurring microbial organism of any one of claims 1-25under conditions and for a sufficient period of time to produce saidcompound of Formula (I).
 27. The method of claim 26, wherein said methodfurther comprises separating the compound of Formula (I) from othercomponents in the culture.
 28. The method of claim 27, wherein theseparating comprises extraction, continuous liquid-liquid extraction,pervaporation, membrane filtration, membrane separation, reverseosmosis, electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, absorptionchromatography, or ultrafiltration.
 29. Culture medium comprisingbioderived compound of Formula (I):

wherein R₁ is C₁₋₂₄ linear alkyl; R₂ is CH₂OH, CHO, or COOH; R₃ is H,OH, or oxo (═O); and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four, wherein said bioderivedcompound has a carbon-12, carbon-13 and carbon-14 isotope ratio thatreflects an atmospheric carbon dioxide uptake source.
 30. The culturemedium of claim 29, wherein said culture medium is separated from anon-naturally occurring microbial organism having a malonyl-CoAindependent fatty acyl-CoA elongation (MI-FAE) cycle and/or amalonyl-CoA dependent fatty acyl-CoA elongation (MD-FAE) cycle incombination with a termination pathway.
 31. A bioderived compound ofFormula (I):

wherein R₁ is C₁₋₂₄ linear alkyl; R₂ is CH₂OH, CHO, or COOH; R₃ is H,OH, or oxo (═O); and

represents a single or double bond with the proviso that the valency ofthe carbon atom to which R₃ is attached is four, having a carbon-12,carbon-13 and carbon-14 isotope ratio that reflects an atmosphericcarbon dioxide uptake source.
 32. The bioderived compound of claim 31,wherein said bioderived compound has an Fm value of at least 80%, atleast 85%, at least 90%, at least 95% or at least 98%.
 33. Thebioderived compound produced according to the method of any one ofclaims 26-28.
 34. A composition comprising said bioderived compound ofany one of claims 31-33 and a compound other than said bioderivedcompound.
 35. The composition of claim 34 wherein said compound otherthan said bioderived compound is a trace amount of a cellular portion ofa non-naturally occurring microbial organism having a malonyl-CoAindependent fatty acyl-CoA elongation (MI-FAE) cycle and/or amalonyl-CoA dependent fatty acyl-CoA elongation (MD-FAE) cycle incombination with a termination pathway.
 36. A composition comprising thebioderived compound of any one of claims 31-33, or a cell lysate orculture supernatant thereof.
 37. A biobased product comprising saidbioderived compound of any one of claims 31-33, wherein said biobasedproduct is a biofuel, chemical, polymer, surfactant, soap, detergent,shampoo, lubricating oil additive, fragrance, flavor material oracrylate.
 38. The biobased product of claim 37 comprising at least 5%,at least 10%, at least 20%, at least 30%, at least 40% or at least 50%said bioderived compound.
 39. The biobased product of claim 37 or 38,wherein said biobased product comprises a portion of said bioderivedcompound as a repeating unit.
 40. A molded product obtained by molding abiobased product of any one of claims 37-39, biobased product is apolymer.
 41. A process for producing a biobased product of any one ofclaims 37-39 comprising chemically reacting said bioderived compoundwith itself or another compound in a reaction that produces saidbiobased product.